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Loss of NF1 plays a major role as an oncogenic driver in many cancer types and can be found in up to 33% of all breast cancers (BC). Loss of NF1 is also a prognostic indicator for increased cancer risk at an earlier age, poorer outcomes, and therapeutic resistance. In addition, certain NF1 genotypes may increase cancer risks, while others do not. NF1 is largely perceived as a classic Ras-opathy syndrome due to inactivating mutations in neurofibromin affecting RAS-MAPK signaling. However, recently it has been shown that NF1 binds estrogen receptor alpha (ER) and acts as a transcriptional corepressor. This helps explain some BC findings specifically in ER+ BC. In this model, specific changes to NF1 that abrogate ER signaling lead to Ras driven tumor resistance to endocrine therapy as cells are able to grow in low levels of E2 (and tamoxifen). Hence, NF1-mutant tumors represent a distinct molecular class in need of new therapeutics. A preclinical mammalian model of NF1 loss and BC would be helpful in both evaluating the role of NF1/ER transcriptional signaling in BC, evaluating the role of immune cells in BC, and testing therapeutics. Novel Nf1 rat models have a very robust ER+ BC phenotype, therefore more closely recapitulating clinical tumors compared to other preclinical models. Our models include a pathogenic patient missense allele c.3827G>A, p.R1276Q (knockin or KI), associated in humans with familial spinal NF1 and malignancy, as well as a 14 base pair deletion c.3661_3674del, p.P1220fs*1223 (knockout or KO) model. Heterozygous (het) Nf1 females develop mammary gland adenocarcinoma spontaneously, but het KO rats develop multiple tumors with earlier onset while het KI rats tend to develop fewer tumors with later onset. Tumors are generally Grade 2 and do not differ by genotype. By 16 weeks, 70% of KO females have developed at least one tumor whereas only 20% of KI rats and 2% of WT rats have developed tumors. This impacts survival as by 1 year 76% of KI females survive yet only 58% of KO females survive. However, by 2 years, both alleles have 54% survival. The divergence in phenotype between patient and null alleles may be due to residual function of R1276Q missense NF1 protein. A more in-depth analysis indicates that mammary tumor formation likely begins relatively early, as we find evidence of aberrant morphology and hyperplasia prior to the formation of palpable tumors. Interestingly, we find histological evidence of lung metastases and expression of breast markers GCDFP15, MGA, and CEA in the lung. Again, we see allele-specific effects in that KO rats develop lung tumors earlier than KI rats. While het male rats also develop mammary tumors at low rates, they experience longer survival times (76% of KO males survive 1 year and 98% of KI males survive 1 year) and males also develop tumors in other locations. Using single nuclei RNASeq to characterize the transcriptional profile of the mammary tumors, we find allele-specific effects beyond repression of Ras activity that drive aggressive tumor development. We identified different tumor cell populations (2 epithelial cell populations, Myeloid cells, B cells, T cells, Basal mammary cells, and WT specific cells) and identified different pathways altered due to the loss of Nf1 including Ephrin B signaling, Cyclin and Cell Cycle signaling, and Glycolysis signaling. Our overall goal is to characterize the phenotype of these rat models in terms of histopathology, Ras signaling, hormone signaling, immune components, and targeted drug response and compare/contrast them with what is known regarding patients with somatic or germline inactivation of NF1 and breast cancer. Ultimately, this will provide better prognostic predictions for patients and better therapeutic options for treatment. Citation Format: Deeann Wallis, Christian Fay, Kelley Bradley, Erik Westin, William Bradley, Hui Liu, Laura Lambert, David Crossman, Jeremy Foote, Robert Kesterson. Loss of NF1 drives hormone dependent mammary carcinogenesis in a rat model with intact immune system [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-08-01.

Supplementary Figure 2 from Growth Factor Independence-1 Is Expressed in Primary Human Neuroendocrine Lung Carcinomas and Mediates the Differentiation of Murine Pulmonary Neuroendocrine Cells

Supplementary Figure 1 from Growth Factor Independence-1 Is Expressed in Primary Human Neuroendocrine Lung Carcinomas and Mediates the Differentiation of Murine Pulmonary Neuroendocrine Cells

Human small cell lung cancers might be derived from pulmonary cells with a neuroendocrine phenotype. They are driven to proliferate by autocrine and paracrine neuropeptide growth factor stimulation. The molecular basis of the neuroendocrine phenotype of lung carcinomas is relatively unknown. The Achaete-Scute Homologue-1 (ASH1) transcription factor is critically required for the formation of pulmonary neuroendocrine cells and is a marker for human small cell lung cancers. The Drosophila orthologues of ASH1 (Achaete and Scute) and the growth factor independence-1 (GFI1) oncoprotein (Senseless) genetically interact to inhibit Notch signaling and specify fly sensory organ development. Here, we show that GFI1, as with ASH1, is expressed in neuroendocrine lung cancer cell lines and that GFI1 in lung cancer cell lines functions as a DNA-binding transcriptional repressor protein. Forced expression of GFI1 potentiates tumor formation of small-cell lung carcinoma cells. In primary human lung cancer specimens, GFI1 expression strongly correlates with expression of ASH1, the neuroendocrine growth factor gastrin-releasing peptide, and neuroendocrine markers synaptophysin and chromogranin A (P < 0.0000001). GFI1 colocalizes with chromogranin A and calcitonin-gene–related peptide in embryonic and adult murine pulmonary neuroendocrine cells. In addition, mice with a mutation in GFI1 display abnormal development of pulmonary neuroendocrine cells, indicating that GFI1 is important for neuroendocrine differentiation.

We have created a panel of 29 NF1 variant complementary DNAs (cDNAs) representing missense variants, many with clinically relevant phenotypes, in-frame deletions, splice variants, and nonsense variants. We have determined the functional consequences of the variants, assessing their ability to produce mature neurofibromin and restore Ras signaling activity in NF1 null (-/-) cells. cDNAs demonstrate variant-specific differences in neurofibromin protein levels, suggesting that some variants lead to neurofibromatosis type 1 (NF1) gene or protein instability or enhanced degradation. When expressed at high levels, some variant proteins are still able to repress Ras activity, indicating that the NF1 phenotype may be due to low protein abundance. In contrast, other variant proteins are incapable of repressing Ras activity, indicating that some do not functionally engage Ras and stimulate GTPase activity. We observed that effects on protein abundance and Ras activity can be mutually exclusive. These assays allow us to categorize variants by functional effects, may help to classify variants of unknown significance, and may have future implications for more directed therapeutics.

ObjectiveTo summarize existing biomarker data for cutaneous neurofibroma (cNF) and to inform the incorporation of biomarkers into clinical trial design for cNFs.MethodsThe cNF working group, a subgroup of the Response Evaluation in Neurofibromatosis and Schwannomatosis (REiNS) consortium, was formed to review and inform clinical trial design for cNFs. Between June 2018 and February 2020, the cNF working group performed a review of existing data on genetic biomarkers for cNFs in the setting of neurofibromatosis type 1. We also reviewed criteria for successful biomarker application in the clinic. The group then held a series of meetings to develop a consensus report.ResultsOur systematic literature review of existing data revealed a lack of validated biomarkers for cNFs. In our report, we summarize the existing signaling, genomic, transcriptomic, histopathologic, and proteomic data relevant to cNF. Finally, we make recommendations for incorporating exploratory aims for predictive biomarkers into clinical trials through biobanking samples.ConclusionThese recommendations are intended to provide both researchers and clinicians with best practices for clinical trial design to aid in the identification of clinically validated biomarkers for cNF.

Loss of NF1 plays a major role as an oncogenic driver in up to 33% of all breast cancers (BC). Loss of NF1 is also a prognostic indicator for increased cancer risk at an earlier age, poorer outcomes, and therapeutic resistance. NF1 has recently been shown to bind the estrogen receptor alpha (ER) and act as a transcriptional corepressor. This helps explain findings in ER+ BC. Changes to NF1 that abrogate ER signaling thereby lead to Ras driven tumor resistance to endocrine therapy as cells are able to grow in low levels of E2 (and tamoxifen). Hence, NF1-mutant tumors represent a distinct molecular class in need of new therapeutics targeting both Ras and ER pathways. We have generated preclinical mammalian models of NF1 loss and BC that can be used to evaluate the role of NF1/ER transcriptional signaling in BC, the role of immune cells in BC, and therapeutics. Our models include a pathogenic patient missense allele c.3827G>A, p.R1276Q (knockin or KI) as well as a 14 base pair deletion c.3661_3674del, p.P1220fs*1223 (knockout or KO) model. Heterozygous (het) Nf1 female rats develop mammary gland adenocarcinoma spontaneously, but het KO rats develop multiple tumors with earlier onset. Tumors are generally Grade 2 and do not differ by genotype. By 16 weeks, 70% of KO females have developed at least one tumor whereas only 20% of KI rats and 2% of WT rats have developed tumors. This impacts survival as 76% of KI and 58% of KO females survive to one year. By 2 years, both alleles have 54% survival. The divergence in phenotype between KI and KO alleles may be due to residual function of R1276Q missense NF1 protein. A more in-depth analysis indicates that mammary tumor formation likely begins relatively early, as we find evidence of aberrant morphology and hyperplasia prior to the formation of palpable tumors. Interestingly, we find histological evidence of lung metastases and expression of breast markers GCDFP15, MGA, and CEA in the lung. Again, we see allele-specific effects in that KO rats develop lung tumors earlier than KI rats. Using single nuclei RNASeq to characterize the transcriptional profile of the mammary tumors, we find allele-specific effects beyond repression of Ras activity that drive aggressive tumor development. We identified different tumor cell populations (2 epithelial cell populations, Myeloid cells, B cells, T cells, Basal mammary cells, and WT specific cells) and identified different pathways altered due to the loss of Nf1 including Ephrin B signaling, Cyclin and Cell Cycle signaling, and Glycolysis signaling. Our overall goal is to characterize the phenotype of these rat models in terms of histopathology, Ras signaling, hormone signaling, immune components, and targeted drug response; which can then be compared with what is known regarding patients with somatic or germline inactivation of NF1 and breast cancer. Ultimately, this will provide better prognostic predictions for patients and better therapeutic options. Citation Format: Deeann Wallis, Christian Fay, Kelley Bradley, Erik Westin, William Bradley, Hui Liu, David Crossman, Jeremy B. Foote, Robert Kesterson. Loss of NF1 drives hormone dependent mammary carcinogenesis in a rat model with intact immune system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1726.

Significant advances in biotechnology have led to the development of a number of different mutation-directed therapies. Some of these techniques have matured to a level that has allowed testing in clinical trials, but few have made it to approval by drug-regulatory bodies for the treatment of specific diseases. While there are still various hurdles to be overcome, recent success stories have proven the potential power of mutation-directed therapies and have fueled the hope of finding therapeutics for other genetic disorders. In this review, we summarize the state-of-the-art of various therapeutic approaches and assess their applicability to the genetic disorder neurofibromatosis type I (NF1). NF1 is caused by the loss of function of neurofibromin, a tumor suppressor and downregulator of the Ras signaling pathway. The condition is characterized by a variety of phenotypes and includes symptoms such as skin spots, nervous system tumors, skeletal dysplasia, and others. Hence, depending on the patient, therapeutics may need to target different tissues and cell types. While we also discuss the delivery of therapeutics, in particular via viral vectors and nanoparticles, our main focus is on therapeutic techniques that reconstitute functional neurofibromin, most notably cDNA replacement, CRISPR-based DNA repair, RNA repair, antisense oligonucleotide therapeutics including exon skipping, and nonsense suppression., Graphical Abstract, We present a comprehensive review that addresses therapeutic approaches to treating specific genetic defects that cause neurofibromatosis type I, including nanoparticle delivery, exon skipping, gene editing, mRNA trans-splicing ribozymes, and non-sense suppression therapeutics. Our multi-faceted approach can be utilized for virtually any rare disease to affect personalized medicine.

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder with almost 3000 different disease-causing variants within the NF1 gene identified. Up to 44% of these variants cause splicing errors to occur within pre-mRNA. A recurrent variant in exon 13, c.1466A>G; p.Y489C (Y489C) results in the creation of an intragenic cryptic splice site, aberrant splicing, a 62 base pair deletion from the mRNA, and subsequent frameshift. We investigated the ability of phosphorodiamidate morpholino oligomers (PMOs) to mask this variant on the RNA level, thus restoring normal splicing. To model this variant, we have developed a human iPS cell line homozygous for the variant using CRISPR/Cas9. PMOs were designed to be 25 base pairs long, and to cover the mutation site so it could not be read by splicing machinery. Results from our in vitro testing showed restoration of normal splicing in the RNA and restoration of full length neurofibromin protein. In addition, we observe the restoration of neurofibromin functionality through GTP-Ras and pERK/ERK testing. The results from this study demonstrate the ability of a PMO to correct splicing errors in NF1 variants at the RNA level, which could open the door for splicing corrections for other variants in this and a variety of diseases.

Variants within the Neurotrophic Tyrosine Kinase Receptor Type 2 (NTRK2) gene have been discovered to play a role in developmental and epileptic encephalopathies, a group of debilitating conditions for which little is known about cause or treatment. Here, we determine the functional consequences of two variants: p.Tyr434Cys (Y434C) (located in the transmembrane domain) and p.Thr720Ile (T720I) (located in the catalytic domain). Wild-type and variant cDNAs were constructed and transfected into HEK293 cells. In cell culture, variant Y434C exhibited ligand-independent activation of tropomyosin-related kinase B (TRKB) signaling with an associated abnormal response to brain-derived neurotrophic factor (BDNF) stimulation and increased levels of phosphorylated extracellular signal-regulated kinase (ERK) and ETS like-1 protein (ELK1) activity. Expression of variant T720I resulted in decreased TRKB signaling with reduced mTor activity as determined by decreased levels of phosphorylated S6. With the deleterious mechanisms characterized, we utilized mediKanren (a novel artificial intelligence tool) to identify therapeutics to compensate for the pathological effects. Downregulation of TRKB through inhibition with mediKanren-predicted compound 1NM-PP1 led to decreased MEK activity. Upregulation of TRKB signaling by mediKanren-predicted valproic acid led to subsequent increase of mTor activity. Overall, our results provide further characterization of the pathogenicity of these two variants in the NTRK2 gene. Indeed, Y434C is the first patient-specific NTRK2 variant with demonstrated hypermorphic activity. Furthermore, we observed that variants Y434C and T720I result in distinct functional consequences that require distinct therapeutic strategies. These data suggest the possibility that unique mutations within different regions of the NTRK2 gene results in separate clinical presentations, representing distinct genetic disorders requiring unique therapeutics.

We investigated the feasibility of utilizing an exon-skipping approach as a genotype-dependent therapeutic for neurofibromatosis type 1 (NF1) by determining which

We investigated the feasibility of utilizing an exon skipping approach as a genotype-dependent therapeutic for neurofibromatosis type 1 (NF1) by determining which NF1 exons might be skipped while maintaining neurofibromin function. Human neurofibromin is well-known as a GTPase activating protein (GAP), but outside of its GAP-related domain (GRD), it is unclear how critical other regions are for function. Initial in silico analysis predicted exons that can be skipped with minimal loss of neurofibromin function. Utilizing a novel Nf1 cDNA system, we performed a functional screen to determine the effects of exon skipping on in vitro neurofibromin expression and GRD function. Loss of single exons 12, 17, 25, 41, 47, or 52 maintained significant GRD function in at least two Ras activity assays. Exons 18/19, 20 and 28 are critical for GRD function; deletion of exons 20, 41, or 47 led to significantly lower levels of neurofibromin. As suggested by in silico analysis, skipping of exons 17 or 52 resulted in both the highest neurofibromin levels and the greatest suppression of Ras activity. Assessment of NF1 patient databases indicates that pathogenic variants resulting in deletion or skipping of exons 17, 25, and 52 have not been reported; and truncating pathogenic variants in each exon account for ~0.91, 0.94, and 0.25% of unrelated NF1 cases, respectively. Hence, we designed antisense phosphodiamitate morpholino oligos (PMOs) to skip exon 17 and evaluated them in human cell lines that we generated via CRISPR/Cas9 with a patient-specific truncating pathogenic variant, c.1885G>A. We down-selected oligos that efficiently caused skipping of exon 17 and restored NF1 expression and function. Further, homozygous deletion of exon 17 in a novel mouse model is compatible with viable and grossly healthy animals with normal lifespan and no tumor development, providing proof-of-concept that exon 17 is not essential for murine neurofibromin function. Mild phenotypes observed include abnormal nesting behavior and lymphoid hyperplasia with increased numbers of both B- and T-cells. Hence, exon skipping should be further investigated as a therapeutic approach for NF1 patients with treatment of individuals with pathogenic variants in exon 17.

Introduction: We have discovered novel non-toxic rifamycins that are extremely potent at sensitizing many drug-resistant cancers, including DLBCL, to standard-of-care chemotherapeutics. Methods: CHOP-resistant DLBCL cell lines were derived from CHOP-sensitive cells by “on-off” cycles of CHOP treatment, analogous to clinical therapy. The CHOP-resistant cells were used in high-throughput screening of a highly diverse collection of approximately 50,000 drug-like molecules to identify small molecules that reverse CHOP-resistance. The FDA-approved drug, Rifabutin, was identified as a non-cytotoxic compound that potently reversed resistance to CHOP. Structure-Activity-Relationships (SAR) on Rifabutin were conducted, which led to the generation of a new more potent CHOP-chemosensitizing agent, designated RTI. Results: RTI was highly synergistic with variety of chemotherapeutics, including doxorubicin (DOX), epirubicin, vinblastine, etoposide in drug-resistant NHL cells. Combination therapy of DOX+RTI in mouse xenograft models of DLBCL was much more effective at repressing tumor growth than with DOX alone. RTI lowered the IC90 in a dose-dependent manner in CD20-positive B cells in bone marrow aspirates from both CHOP-naïve and CHOP-relapsed patients, and in a metastatic lymph node. RTI’s PK characteristics are similar to rifabutin, and it exhibited no overt toxicity in mice or pigs at high doses. RTI rapidly induced mitochondrial superoxide, membrane potential, and fission. The superoxide dismutase, FeTCP, antagonized RTI-induced ROS and potentiation of DOX cytotoxicity. RTI reduced the activity of the Nrf-2 antioxidant protein, upregulated proteins involved in the unfolded protein response (UPR), and induced metabolic reprogramming as indicated by Seahorse assays that showed increased glycolysis and decreased oxygen consumption from 3 to 24 hrs. Conclusions: RTI has a broad spectrum of action in both double- and triple-hit DLBCL and synergizes with many different chemotherapeutics to restore drug sensitivity. RTI-79 works by dramatically increasing intracellular superoxide through redox cycling, triggers UPR, and downregulates Nrf-2 activity. Thus, RTI-79 increases oxidative stress through the squelching of Nrf-2’s ability to respond to chemotherapeutic stress. Since the parent compound, rifabutin, binds to the aryl hydrocarbon receptor (AhR) and modulates its activity, we hypothesize that RTI mediates its unique pleiotropic chemosensitizing mechanism through targeting of AhR, and provides for a broad, safe, and novel approach to treating drug resistant cancers. Ongoing experiments are investigating the effect of RTI on the AhR-signaling pathway and the role it plays in RTI-mediated chemosensitization and potentiation of chemotherapeutics. A clinical trial of companion dogs with CHOP-relapsed DLBCL treated with combination CHOP+RTI is currently in progress. Citation Format: Steve A. Maxwell, Deeann Wallis, Nian Zhou, Dwight Baker, Seyed H. Mousavi-Fard, Kimberly Loesch, Stacy Galaviz, Liam Guthrie, Thomas Snavely, Qingan Sun, Carolina M. Rojas, David W. Threadgill, Thomas Ioerger, Wen Dong, Gwen Seemann, Theresa W. Fossum, James C. Sacchettini. Development of novel, non-toxic rifamycins that reverse drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 410.

Neurofibromatosis Type 1 (NF1) is caused by pathogenic variants in the NF1 gene encoding neurofibromin. Definition of NF1 protein&ndash, protein interactions (PPIs) has been difficult and lacks replication, making it challenging to define binding partners that modulate its function. We created a novel tandem affinity purification (TAP) tag cloned in frame to the 3&rsquo, end of the full-length murine Nf1 cDNA (mNf1). We show that this cDNA is functional and expresses neurofibromin, His-Tag, and can correct p-ERK/ERK ratios in NF1 null HEK293 cells. We used this affinity tag to purify binding partners with Strep-Tactin&reg, XT beads and subsequently, identified them via mass spectrometry (MS). We found the tagged mNf1 can affinity purify human neurofibromin and vice versa, indicating that neurofibromin oligomerizes. We identify 21 additional proteins with high confidence of interaction with neurofibromin. After Metacore network analysis of these 21 proteins, eight appear within the same network, primarily keratins regulated by estrogen receptors. Previously, we have shown that neurofibromin levels negatively regulate keratin expression. Here, we show through pharmacological inhibition that this is independent of Ras signaling, as the inhibitors, selumetinib and rapamycin, do not alter keratin expression. Further characterization of neurofibromin oligomerization and binding partners could aid in discovering new neurofibromin functions outside of Ras regulation, leading to novel drug targets.

Factors genètics; Desordre d'ús de substàncies Factores genéticos; Desorden de uso de sustancias Genetic factors; Substance use disorder; ADGRL3 (LPHN3) Genetic factors are strongly implicated in the susceptibility to develop externalizing syndromes such as attention-deficit/hyperactivity disorder (ADHD), oppositional defiant disorder, conduct disorder, and substance use disorder (SUD). Variants in the ADGRL3 (LPHN3) gene predispose to ADHD and predict ADHD severity, disruptive behaviors comorbidity, long-term outcome, and response to treatment. In this study, we investigated whether variants within ADGRL3 are associated with SUD, a disorder that is frequently co-morbid with ADHD. Using family-based, case-control, and longitudinal samples from disparate regions of the world (n = 2698), recruited either for clinical, genetic epidemiological or pharmacogenomic studies of ADHD, we assembled recursive-partitioning frameworks (classification tree analyses) with clinical, demographic, and ADGRL3 genetic information to predict SUD susceptibility. Our results indicate that SUD can be efficiently and robustly predicted in ADHD participants. The genetic models used remained highly efficient in predicting SUD in a large sample of individuals with severe SUD from a psychiatric institution that were not ascertained on the basis of ADHD diagnosis, thus identifying ADGRL3 as a risk gene for SUD. Recursive-partitioning analyses revealed that rs4860437 was the predominant predictive variant. This new methodological approach offers novel insights into higher order predictive interactions and offers a unique opportunity for translational application in the clinical assessment of patients at high risk for SUD R01 DA039881/DA/NIDA NIH HHS/United States. DA039881/U.S. Department of Health & Human Services NIH, National Institute on Drug Abuse (NIDA)/

Background The Latrophilin 3 (LPHN3) gene (recently renamed Adhesion G protein-coupled receptor L3 (ADGRL3)) has been linked to susceptibility to attention deficit/hyperactivity disorder (ADHD) and vulnerability to addiction. However, its role and function are not well understood as there are no known functional variants. Methods To characterize the function of this little known gene, we phenotyped Lphn3 null mice. We assessed motivation for food reward and working memory via instrumental responding tasks, motor coordination via rotarod, and depressive-like behavior via forced swim. We also measured neurite outgrowth of primary hippocampal and cortical neuron cultures. Standard blood chemistries and blood counts were performed. Finally, we also evaluated the transcriptome in several brain regions. Results Behaviorally, loss of Lphn3 increases both reward motivation and activity levels. Lphn3 null mice display significantly greater instrumental responding for food than wild-type mice, particularly under high response ratios, and swim incessantly during a forced swim assay. However, loss of Lphn3 does not interfere with working memory or motor coordination. Primary hippocampal and cortical neuron cultures demonstrate that null neurons display comparatively enhanced neurite outgrowth after 2 and 3 days in vitro. Standard blood chemistry panels reveal that nulls have low serum calcium levels. Finally, analysis of the transcriptome from prefrontal cortical, striatal, and hippocampal tissue at different developmental time points shows that loss of Lphn3 results in genotype-dependent differential gene expression (DGE), particularly for cell adhesion molecules and calcium signaling proteins. Much of the DGE is attenuated with age, and is consistent with the idea that ADHD is associated with delayed cortical maturation. Conclusions Transcriptome changes likely affect neuron structure and function, leading to behavioral anomalies consistent with both ADHD and addiction phenotypes. The data should further motivate analyses of Lphn3 function in the developmental timing of altered gene expression and calcium signaling, and their effects on neuronal structure/function during development.

Background Neurofibromatosis type I (NF1) is caused by heterozygous loss-of-function variants in the NF1 gene encoding neurofibromin which serves as a tumor suppressor that inhibits RAS signaling and regulates cell proliferation and differentiation. While, the only well-established functional domain in the NF1 protein is the GAP-related domain (GRD), most of the identified non-truncating disease-causing variants are located outside of this domain, supporting the existence of other important disease-associated domains. Identifying these domains may reveal novel functions of NF1. Methods By implementing inferential statistics combined with machine-learning methods, we developed a novel NF1-specific functional prediction model that focuses on nonsynonymous single nucleotide variants (SNVs). The model enables annotating all possible NF1 nonsynonymous variants, thus mapping the range of pathogenic non-truncating variants at the codon level across the NF1 gene. Findings The generated model demonstrates high absolute prediction value for missense and splice-site variations (area under the ROC curve of 0.96) outperforming 14 other established models. By reviewing the entire dataset of nonsynonymous variants, two novel domains (Armadillo type fold 1 and 2) were identified as being associated with pathogenicity (OR 1.86; CI 1.04 to 3.34 and OR 2.08; CI 1.08 to 4.04, respectively; P

We tested the hypothesis thatin uteroexposure to heavy metals increases autism-like behavioral phenotypes in adult animals and induces epigenetic changes in genes that have roles in the etiology of autism. Mouse dams were treated with cadmium, lead, arsenate, manganese, and mercury via drinking water from gestational days (E) 1–10. Valproic acid (VPA) injected intraperitoneally once on (E) 8.5 served as a positive control. Young male offspring were tested for behavioral deficits using four standardized behavioral assays. In this study,in uteroexposure to heavy metals resulted in multiple behavioral abnormalities that persisted into adulthood. VPA and manganese induced changes in perseverative/impulsive behavior and social dominance behavior, arsenic caused changes only in perseverative/impulsive behavior, and lead induced abnormalities in social interaction in comparison to the control animals. Brain samples from Mn, Pb, and VPA treated and control animals were evaluated for changes in CpG island methylation in promoter regions and associated changes in gene expression. The Chd7 gene, essential for neural crest cell migration and patterning, was found to be hypomethylated in each experimental animal tested compared to water-treated controls. Furthermore, distinct patterns of CpG island methylation yielded novel candidate genes for further investigation.

Loss of NF1 is an oncogenic driver. In efforts to define pathways responsible for the development of neurofibromas and other cancers, transcriptomic and proteomic changes are evaluated in a non-malignant NF1 null cell line. NF1 null HEK293 cells were created using CRISPR/Cas9 technology and they are compared to parental cells that express neurofibromin. A total of 1222 genes and 132 proteins are found to be differentially expressed. The analysis is integrated to identify eight transcripts/proteins that are differentially regulated in both analyses. Metacore Pathway analysis identifies Neurogenesis NGF/TrkA MAPK-mediated signaling alterations. Next, the data set is compared with other published studies that involve analysis of cells or tumors deficient for NF1 and it is found that 141 genes recur in the sample and others; only thirteen of these genes recur in two or more studies. Genes/proteins of interest are validated via q-RT-PCR or Western blot. It is shown that KRT8 and 14-3-3σ protein levels respond to exogenously introduced mNf1 cDNA. Hence, transcripts/proteins that respond to neurofibromin levels are identified and they can potentially be used as biomarkers.

The LPHN3 gene has been associated with both attention deficit-hyperactivity disorder (ADHD) and addiction, suggesting that it may play a role in the etiology of these disorders. Unfortunately, almost nothing is known about the normal functions of this gene, which has hampered understanding of its potential pathogenic role. To begin to characterize such normal functions, we utilized a gene-trap embryonic stem cell line to generate mice mutant for the Lphn3 gene. We evaluated differential gene expression in whole mouse brain between mutant and wild type male littermates at postnatal day 0 using TaqMan gene expression assays. Most notably, we found changes in dopamine and serotonin receptors and transporters (Dat1, Drd4, 5Htt, 5Ht2a), changes in neurotransmitter metabolism genes (Th, Gad1), as well as changes in neural developmental genes (Nurr, Ncam). When mice were examined at 4–6 weeks of age, null mutants showed increased levels of dopamine and serotonin in the dorsal striatum. Finally, null mutant mice had a hyperactive phenotype in the open field test, independent of sex, and were more sensitive to the locomotor stimulant effects of cocaine. Considered together, these results suggest that Lphn3 plays a role in development and/or regulation of monoamine signaling. Given the central role for monoamines in ADHD and addiction, it seems likely that the influence of LPHN3 genotype on these disorders is mediated through alterations in monoamine signaling.

Elucidating the genetic determinants of radiation response is crucial to optimizing and individualizing radiotherapy for cancer patients. In order to identify genes that are involved in enhanced sensitivity or resistance to radiation, a library of stable mutant murine embryonic stem cells (ESCs), each with a defined mutation, was screened for cell viability and gene expression in response to radiation exposure. We focused on a cancer-relevant subset of over 500 mutant ESC lines. We identified 13 genes; 7 genes that have been previously implicated in radiation response and 6 other genes that have never been implicated in radiation response. After screening, proteomic analysis showed enrichment for genes involved in cellular component disassembly (e.g. Dstn and Pex14) and regulation of growth (e.g. Adnp2, Epc1, and Ing4). Overall, the best targets with the highest potential for sensitizing cancer cells to radiation were Dstn and Map2k6, and the best targets for enhancing resistance to radiation were Iqgap and Vcan. Hence, we provide compelling evidence that screening mutant ESCs is a powerful approach to identify genes that alter radiation response. Ultimately, this knowledge can be used to define genetic variants or therapeutic targets that will enhance clinical therapy.

Background Turner syndrome (TS) results from the loss of part or all of one X chromosome in females. It can result in short stature, various dysmorphic findings, and difficulties with psychosocial adjustment. Girls with TS have previously been found to exhibit increased levels of hyperactivity and inattention. However, no studies have assessed whether individuals with TS meet strict (DSM-IV) criteria for attention-deficit/hyperactivity disorder (ADHD). Objective We looked at the prevalence of ADHD in girls with TS and evaluated the contribution of imprinting on cognitive performance (IQ) and ADHD. Methods We tested 50 girls with TS for ADHD, IQ, academic performance, and parental origin of the X chromosome. Results We report an 18-fold increase in the prevalence of ADHD in girls with TS (24%) compared with girls in the general population (1.3%) (p < .01) and a 4.8 fold increase when compared with boys and girls in the general population (5%) (p < .05). In contrast to previous reports, our molecular studies in females with 45,X also showed no association between IQ scores and the parental origin of the intact X chromosome. Conclusions We find an increased prevalence of ADHD in girls with TS but no evidence for imprinting effects for cognitive performance.

Gfi1 is a transcriptional repressor implicated in lymphomagenesis, neutropenia, and hematopoietic development, as well as ear and lung development. Here, we demonstrate that Gfi1 functions downstream of Math1 in intestinal secretory lineage differentiation. Gfi1-/- mice lack Paneth cells, have fewer goblet cells, and supernumerary enteroendocrine cells. Gfi1-/- mice show gene expression changes consistent with this altered cell allocation. These data suggest that Gfi1 functions to select goblet/Paneth versus enteroendocrine progenitors. We propose a model of intestinal cell fate choice in which β-catenin and Cdx function upstream of Math1, and lineage-specific genes such as Ngn3 act downstream of Gfi1.

We used a genomic library of mutant murine embryonic stem cells (ESCs) and report the methodology required to simultaneously culture, differentiate, and screen more than 3,200 heterozygous mutant clones to identify host-based genes involved in both sensitivity and resistance to rabies virus infection. Established neuronal differentiation protocols were miniaturized such that many clones could be handled simultaneously, and molecular markers were used to show that the resultant cultures were pan-neuronal. Next, we used a green fluorescent protein (GFP) labeled rabies virus to develop, validate, and implement one of the first host-based, high-content, high-throughput screens for rabies virus. Undifferentiated cell and neuron cultures were infected with GFP-rabies and live imaging was used to evaluate GFP intensity at time points corresponding to initial infection/uptake and early and late replication. Furthermore, supernatants were used to evaluate viral shedding potential. After repeated testing, 63 genes involved in either sensitivity or resistance to rabies infection were identified. To further explore hits, we used a completely independent system (siRNA) to show that reduction in target gene expression leads to the observed phenotype. We validated the immune modulatory gene Unc13d and the dynein adapter gene Bbs4 by treating wild-type ESCs and primary neurons with siRNA; treated cultures were resistant to rabies infection/replication. Overall, the potential of such in vitro functional genomics screens in stem cells adds additional value to other libraries of stem cells. This technique is applicable to any bacterial or virus interactome and any cell or tissue types that can be differentiated from ESCs. Stem Cells 2015;33:2509–2522

Bacteria that cause disease rely on their ability to counteract and overcome host defenses. Here we present a genome-scale study of Mycobacterium tuberculosis (Mtb) that uncovers the bacterial determinants of surviving host immunity, sets of genes we term “counteractomes.” Through this, we find that CD4 T cells attempt to starve Mtb of tryptophan through a mechanism that limits Chlamydia and Leishmania infections. In those cases, tryptophan starvation works well, since those pathogens are natural tryptophan auxotrophs. Mtb, however, can synthesize tryptophan, and thus starvation fails as an Mtb-killing mechanism. We then describe a small molecule inhibitor of Mtb tryptophan synthesis, which turns Mtb into a tryptophan auxotroph and restores the efficacy of a failed host defense. Together, our findings demonstrate that the Mtb determinants for surviving host immunity—Mtb’s immune counteractomes—serve as probes of host immunity, uncovering immune-mediated stresses that can be leveraged for therapeutic discovery.

A cDNA encoding a new human actin-related protein (ARP) was cloned. The corresponding protein is highly conserved with the previously described ARP3 protein, suggesting that it represents a second isoform of the human ARP3 subfamily. This new actin-related protein was subsequently named ARP3β and represents the second example of multiple isoforms of an actin-related protein in a single organism. The ARP3β gene was mapped to chromosome band 7q34, centromeric to Sonic Hedgehog. Gene structure analysis revealed that at least part of the observed ARP3β mRNA heterogeneity is caused by alternative splicing resulting in exon skipping. Transcripts produced after exon 2 skipping are predicted to encode truncated products, whose functionality is still unclear. An ARP3β pseudogene was detected on chromosome 2p11 by database searching. Several ARP3β mRNA species were detected by Northern blotting and their abundance varied importantly among tissues: the highest expression levels were detected in fetal and adult brain, whereas lower levels were observed in liver, muscle and pancreas. In contrast, ARP3 mRNAs were detected in all tissues tested. Using in situ hybridization, the expression of ARP3β in brain was shown to be restricted to neurons and epithelial cells from choroid plexus. This suggests a specific function for ARP3β in the physiology of the development and/or maintenance of distinct subsets of nerve cells.

Holoprosencephaly (HPE) is the most common developmental defect of the forebrain and midface in humans. In holoprosencephaly the cerebral hemispheres of the brain fail to separate into distinct left and right hemispheres. This malformation is due to the improper specification and formation of the forebrain during early development. When one considers the great number and kinds of genetic interactions that must occur to properly pattern the developing forebrain, it is not surprising that HPE is extremely heterogeneous. In addition to teratogenic agents, several genes are implicated as the cause of HPE. At least 12 different loci have been associated with HPE and now several distinct human genes for holoprosencephaly have been identified. These genes include Sonic Hedgehog (SHH), ZIC2, SIX3, and TG-interacting factor (TGIF). Here we present an overview of the presently known genes causing human holoprosencephaly. We discuss their functional role in development of the forebrain and summarize the mutations and polymorphisms that have been identified within them. Hum Mutat 16:99-108, 2000. Published 2000 Wiley-Liss, Inc.

Holoprosencephaly (HPE) is the most common developmental defect of the forebrain in humans. Several distinct human genes for holoprosencephaly have now been identified. They include Sonic hedgehog (SHH), ZIC2, and SIX3. Many additional genes involved in forebrain development are rapidly being cloned and characterized in model vertebrate organisms. These include Patched (Ptc), Smoothened (Smo), cubitus interuptus (ci)/Gli, wingless (wg/Wnt, decapentaplegic (dpp)/BMP, Hedgehog interacting protein (Hip), nodal, Smads, One-eyed pinhead (Oep), and TG-Interacting Factor (TGIF). However, further analysis is needed before their roles in HPE can be established. Here we present an overview of the presently known genes causing human holoprosencephaly and describe candidate genes involved in forebrain development identified in other systems. A model is discussed for how these genes may interact within and between several different signaling pathways to direct the formation of the forebrain.

Drag-swab samples were collected from 18 poultry houses at 9 broiler farms. Fifty drag-swab samples were tested for Salmonella by microbiologic culture using selective enrichment and the polymerase chain reaction (PCR) with oligonucleotide primers specific for all members of the genus Salmonella. Drag-swab samples were tested for Salmonella using PCR before and after enrichment. Only one sample was positive by PCR prior to enrichment. Forty-seven of the drag-swabs samples tested after enrichment were positive for Salmonella using PCR, and 29 were positive by microbiologic culture. All but one of the culture-positive samples were positive by PCR; this discordant sample was classified as indeterminate by PCR. Salmonella was identified in houses from all nine farms by PCR and eight of nine farms by microbiologic culture. Salmonella was found in all 18 houses by PCR and in 15 of 18 houses by microbiologic culture. In this study, PCR was significantly (P < .001) more sensitive than culture for environmental monitoring of Salmonella using drag-swabs.

Salmonella enteritidis was identified in feces from hens using the polymerase chain reaction (PCR) and oligonucleotide primers specific for all members of the genus Salmonella. Feces from specific-pathogen-free Leghorn hens were determined to be negative for Salmonella by microbiological culture and by the PCR. Fecal samples were inoculated with known numbers of colony-forming units of S. enteritidis. The DNA was extracted from fecal samples and amplified by the PCR using genus-specific primers. Salmonella were detected in all samples known to be positive; the sensitivity of the assay extended to 1 cfu of S. enteritidis/ g feces. Feces that were not inoculated with Salmonella were negative. Microbiological culture was less sensitive than the PCR assay; results of culture of feces with less than 102 cfu/g were negative. Although S. enteritidis was used in this study, the oligonucleotide primers used in this study have been previously demonstrated to be genus-specific for Salmonella.

The main characteristic of attention deficit/hyperactivity disorder (ADHD) is a persistent pattern of inattention and/or hyperactivity-impulsivity which is more frequent and severe than is usually expected in individuals at a comparable level of development. ADHD is estimated to affect approximately 5.29% of school-aged children and is therefore the most common childhood onset psychological disorder. A conservative estimate of the annual societal cost of illness for ADHD in childhood and adolescence is USD 42.5 billion in the U.S. alone. Global sales of ADHD medicines could reach USD 4.3 billion by 2012. Despite the prevalence, high heritability and costs of ADHD, biological markers do not exist. Such biomarkers are in high demand as they would help eliminate the subjective diagnoses based on interviews and potentially allow for earlier diagnosis and personalized medicine. Lack of markers likely stems from several factors that complicate ADHD research and the assessment of pharmacological responses. This review analyses complicating factors in defining ADHD phenotype and etiology, identifying specific diagnostic markers and the difficulties in the assessment of pharmacogenomic markers. The dopamine transporter (DAT1) genotype and methylphenidate (MPH) response are detailed as an example of a biomarker. A recent report of a novel ADHD gene and its possible role as a biomarker is explored. Finally, suggestions for strategies and study designs for future research for the definition of effective ADHD biomarkers are made.

Attention-Deficit/Hyperactivity Disorder (ADHD) has a very high heritability (0.8), suggesting that about 80% of phenotypic variance is due to genetic factors. We used the integration of statistical and functional approaches to discover a novel gene that contributes to ADHD. For our statistical approach, we started with a linkage study based on large multigenerational families in a population isolate, followed by fine mapping of targeted regions using a family-based design. Family- and population-based association studies in five samples from disparate regions of the world were used for replication. Brain imaging studies were performed to evaluate gene function. The linkage study discovered a genome region harbored in the Latrophilin 3 gene (LPHN3). In the world-wide samples (total n6360, with 2627 ADHD cases and 2531 controls) statistical association of LPHN3 and ADHD was confirmed. Functional studies revealed that LPHN3 variants are expressed in key brain regions related to attention and activity, affect metabolism in neural circuits implicated in ADHD, and are associated with response to stimulant medication. Linkage and replicated association of ADHD with a novel non-candidate gene (LPHN3) provide new insights into the genetics, neurobiology, and treatment of ADHD. Fil: Arcos Burgos, M.. National Human Genome Research Institute; Estados Unidos Fil: Jain, M.. National Human Genome Research Institute; Estados Unidos Fil: Acosta, M. T.. National Human Genome Research Institute; Estados Unidos Fil: Shively, S.. National Human Genome Research Institute; Estados Unidos. National Institute Of Neurological Disorders And Stroke; Estados Unidos Fil: Stanescu, H.. National Human Genome Research Institute; Estados Unidos Fil: Wallis, D.. National Human Genome Research Institute; Estados Unidos Fil: Domene, Sabina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. National Human Genome Research Institute; Estados Unidos Fil: Vélez, J .I.. National Human Genome Research Institute; Estados Unidos Fil: Karkera, J. D.. National Human Genome Research Institute; Estados Unidos Fil: Balog, J.. National Human Genome Research Institute; Estados Unidos Fil: Berg, K.. National Human Genome Research Institute; Estados Unidos Fil: Kleta, R.. National Human Genome Research Institute; Estados Unidos Fil: Gahl, W. A.. National Human Genome Research Institute; Estados Unidos Fil: Roessler, E.. National Human Genome Research Institute; Estados Unidos Fil: Long, R.. National Human Genome Research Institute; Estados Unidos Fil: Lie, J.. National Institute Of Neurological Disorders And Stroke; Estados Unidos Fil: Pineda, D.. Universidad de Antioquia; Colombia Fil: Londoño, A. C.. Universidad de Antioquia; Colombia Fil: Palacio, J. D.. Universidad de Antioquia; Colombia Fil: Arbelaez, A.. Universidad de Antioquia; Colombia Fil: Lopera, F.. Universidad de Antioquia; Colombia Fil: Elia, J.. The Children's Hospital Of Philadelphia; Estados Unidos Fil: Hakonarson, H.. The Children's Hospital Of Philadelphia; Estados Unidos Fil: Johansson, S.. University Of Bergen; Noruega Fil: Knappskog, P. M.. University Of Bergen; Noruega Fil: Haavik, J.. University Of Bergen; Noruega Fil: Ribases, M.. Hospital Universitari Vall D'hebron; España Fil: Cormand, B.. Universidad de Barcelona; España Fil: Bayes, M.. Universidad de Barcelona; España Fil: Casas, M.. Universitat Autònoma de Barcelona; España. Hospital Universitari Vall D'hebron; España Fil: Ramos Quiroga, J. A.. Universitat Autònoma de Barcelona; España. Hospital Universitari Vall D'hebron; España Fil: Hervas, A.. Hospital Mutua de Terrassa; España Fil: Maher, B. S.. Virginia Institute For Psychiatric And Behavioral Genetics; Estados Unidos Fil: Faraone, S. V.. State University Of New York Upstate Medical University; Estados Unidos Fil: Seitz, C.. Saarland University Hospital; Alemania Fil: Freitag, C. M.. Saarland University Hospital; Alemania Fil: Palmason, H.. University of Trier; Alemania Fil: Meyer, J.. University of Trier; Alemania Fil: Romanos, M.. Universität Würzburg; Alemania Fil: Walitza, S.. Universität Würzburg; Alemania Fil: Hemminger, U.. Universität Würzburg; Alemania Fil: Warnke, A.. Universität Würzburg; Alemania Fil: Romanos, J.. Universität Würzburg; Alemania Fil: Renner, T.. Universität Würzburg; Alemania Fil: Jacob, C.. Universität Würzburg; Alemania Fil: Lesch, K. P.. Universität Würzburg; Alemania Fil: Swanson, J.. University of California at Irvine; Estados Unidos Fil: Vortmeyer, A.. National Human Genome Research Institute; Estados Unidos Fil: Bailey Wilson, J. E.. National Human Genome Research Institute; Estados Unidos Fil: Castellanos, F. X.. University of New York; Estados Unidos Fil: Muenke, M.. National Human Genome Research Institute; Estados Unidos

Objective The intent of this review is to provide an overview for the practicing psychologist/psychiatrist regarding the complexities of and the most recent advances made in the study of the genetic basis of attention-deficit/hyperactivity disorder (ADHD). Methods We review a variety of concepts including: (a) complexities involved in studying the genetics of ADHD, (b) evidence for a primarily genetic component of ADHD, (c) evidence suggesting that there are only a few genes with major effects contributing to ADHD, (d) identification of the best candidate genes, (e) linkage analysis for the identification of novel candidate genes, and (f) data on gene–environment interactions. Results It is now generally accepted that ADHD has a biological and even primarily genetic basis. However, despite the identification of several candidate genes, none of them seems to have a substantial effect and the exact etiology underlying ADHD has remained elusive. Genome-wide linkage analysis can help in the identification of novel candidate genes. While several independent groups have initiated these studies, we await further details and specific genes from fine-mapping studies. Most recently, researchers have been trying to identify gene by environment interactions to help understand ADHD. Replication of positive findings will be essential in teasing out these combinatorial influences. Conclusions Ideally, one day specific genes with major effects and specific risk factors with which they interact will be identified and we will be able to implement personalized medicine. Knowledge of such genes will allow us to identify specific diagnostic biological markers. In addition, defining the target genes is the first step in developing novel drug therapies to treat the ADHD symptoms that lead to impairment. Furthermore, such markers could also identify at risk individuals at a younger age in order to implement treatments sooner to decrease the severity of ADHD symptoms or even to prevent future ADHD symptomatology.

Background Attention-deficit/hyperactivity disorder (ADHD) comorbid with oppositional defiant disorder (ODD) or conduct disorder (CD) and substance abuse/dependence seems to represent a specific subset within the phenotypic ADHD spectrum. Methods We applied complex segregation and linkage analyses in a set of multigenerational families densely segregating ADHD comorbid with ODD, CD, alcohol abuse/dependence, and nicotine dependence. Results Our data suggest that ADHD cosegregates with disruptive behaviors as a unique, phenotypically variable trait as evidenced by highly significant pair-wise linkages among: ADHD and ODD (logarithm of odds [LOD] = 14.19), ADHD and CD (LOD = 5.34), ODD and CD (LOD = 6.68), and CD and alcohol abuse/dependence (LOD = 3.98). In addition to previously reported ADHD susceptibility loci, we found evidence of linkage for comorbid ADHD phenotypes to loci at 8q24, 2p21-22.3, 5p13.1-p13.3, 12p11.23-13.3, 8q15, and 14q21.1-22.2. These results were replicated with an affected status phenotype derived from latent class clusters. Conclusions Patterns of cosegregation of ADHD with comorbidities can inform our understanding of the inheritance patterns not only of ADHD but also of disruptive behavioral disorders and alcohol abuse/dependence. Refining the comorbid ADHD phenotype by determining the cosegregation profile of specific comorbidities might be a powerful tool for defining significant regions of linkage.

Human small cell lung cancers might be derived from pulmonary cells with a neuroendocrine phenotype. They are driven to proliferate by autocrine and paracrine neuropeptide growth factor stimulation. The molecular basis of the neuroendocrine phenotype of lung carcinomas is relatively unknown. The Achaete-Scute Homologue-1 (ASH1) transcription factor is critically required for the formation of pulmonary neuroendocrine cells and is a marker for human small cell lung cancers. The Drosophila orthologues of ASH1 (Achaete and Scute) and the growth factor independence-1 (GFI1) oncoprotein (Senseless) genetically interact to inhibit Notch signaling and specify fly sensory organ development. Here, we show that GFI1, as with ASH1, is expressed in neuroendocrine lung cancer cell lines and that GFI1 in lung cancer cell lines functions as a DNA-binding transcriptional repressor protein. Forced expression of GFI1 potentiates tumor formation of small-cell lung carcinoma cells. In primary human lung cancer specimens, GFI1 expression strongly correlates with expression of ASH1, the neuroendocrine growth factor gastrin-releasing peptide, and neuroendocrine markers synaptophysin and chromogranin A (P < 0.0000001). GFI1 colocalizes with chromogranin A and calcitonin-gene–related peptide in embryonic and adult murine pulmonary neuroendocrine cells. In addition, mice with a mutation in GFI1 display abnormal development of pulmonary neuroendocrine cells, indicating that GFI1 is important for neuroendocrine differentiation.

Gfi1 was first identified as causing interleukin 2-independent growth in T cells and lymphomagenesis in mice. Much work has shown thatGfi1 and Gfi1b, a second mouse homolog, play pivotal roles in blood cell lineage differentiation. However, neither Gfi1 norGfi1b has been implicated in nervous system development, even though their invertebrate homologues, senseless in Drosophila andpag-3 in C. elegans are expressed and required in the nervous system. We show that Gfi1 mRNA is expressed in many areas that give rise to neuronal cells during embryonic development in mouse, and that Gfi1 protein has a more restricted expression pattern. By E12.5Gfi1 mRNA is expressed in both the CNS and PNS as well as in many sensory epithelia including the developing inner ear epithelia. At later developmental stages, Gfi1 expression in the ear is refined to the hair cells and neurons throughout the inner ear. Gfi1 protein is expressed in a more restricted pattern in specialized sensory cells of the PNS, including the eye, presumptive Merkel cells, the lung and hair cells of the inner ear.Gfi1 mutant mice display behavioral defects that are consistent with inner ear anomalies, as they are ataxic, circle, display head tilting behavior and do not respond to noise. They have a unique inner ear phenotype in that the vestibular and ccchlear hair cells are differentially affected. AlthoughGfi1-deficient mice initially specify inner ear hair cells, these hair cells are disorganized in both the vestibule and cochlea. The outer hair cells of the cochlea are improperly innervated and express neuronal markers that are not normally expressed in these cells. Furthermore, Gfi1mutant mice lose all cochlear hair cells just prior to and soon after birth through apoptosis. Finally, by five months of age there is also a dramatic reduction in the number of cochlear neurons. Hence, Gfi1 is expressed in the developing nervous system, is required for inner ear hair cell differentiation, and its loss causes programmed cell death.

Holoprosencephaly (HPE) is a common, severe malformation of the brain that involves separation of the central nervous system into left and right halves. Mild HPE can consist of signs such as a single central incisor, hypotelorism, microcephaly, or other craniofacial findings that can be present with or without associated brain malformations1,2,3. The aetiology of HPE is extremely heterogeneous, with the proposed participation of a minimum of 12 HPE-associated genetic loci as well as the causal involvement of specific teratogens acting at the earliest stages of neurulation4. The HPE2 locus was recently characterized as a 1-Mb interval on human chromosome 2p21 that contained a gene associated with HPE. A minimal critical region was defined by a set of six overlapping deletions and three clustered translocations in HPE patients5. We describe here the isolation and characterization of the human homeobox-containing SIX3 gene from the HPE2 minimal critical region (MCR). We show that at least 2 of the HPE-associated translocation breakpoints in 2p21 are less than 200 kb from the 5´ end of SIX3. Mutational analysis has identified four different mutations in the homeodomain of SIX3 that are predicted to interfere with transcriptional activation and are associated with HPE. We propose that SIX3 is the HPE2 gene, essential for the development of the anterior neural plate and eye in humans.

Salmonella was identified in feces from horses, using the polymerase chain reaction (PCR) and genus-specific oligonucleotide primers. Feces from healthy horses were determined to be culture negative and PCR negative for Salmonella. Fecal samples were inoculated with known numbers of colony-forming units (CFU) of S. enteritidis. The fecal samples were enriched overnight in tetrathionate broth, and then DNA was extracted and amplified by PCR using genus-specific primers. Sensitivity of the assay extended to 100 CFU Salmonella enteritidis/g feces; sensitivity of microbiologic culture with enrichment extended to 100 CFU Salmonella enteritidis/g feces. Feces that were not inoculated with S. enteritidis were negative by the PCR. Detection of salmonellae in feces was possible using the PCR within 24 hours from the time of submission of samples. Because samples were enriched, isolates were available for determining antibiograms and serologic grouping or typing.