Search

BACKGROUND: Many patients remain without a diagnosis despite extensive medical evaluation. The Undiagnosed Diseases Network (UDN) was established to apply a multidisciplinary model in the evaluation of the most challenging cases and to identify the biologic characteristics of newly discovered diseases. The UDN, which is funded by the National Institutes of Health, was formed in 2014 as a network of seven clinical sites, two sequencing cores, and a coordinating center. Later, a central biorepository, a metabolomics core, and a model organisms screening center were added. METHODS: We evaluated patients who were referred to the UDN over a period of 20 months. The patients were required to have an undiagnosed condition despite thorough evaluation by a health care provider. We determined the rate of diagnosis among patients who subsequently had a complete evaluation, and we observed the effect of diagnosis on medical care. RESULTS: A total of 1519 patients (53% female) were referred to the UDN, of whom 601 (40%) were accepted for evaluation. Of the accepted patients, 192 (32%) had previously undergone exome sequencing. Symptoms were neurologic in 40% of the applicants, musculoskeletal in 10%, immunologic in 7%, gastrointestinal in 7%, and rheumatologic in 6%. Of the 382 patients who had a complete evaluation, 132 received a diagnosis, yielding a rate of diagnosis of 35%. A total of 15 diagnoses (11%) were made by clinical review alone, and 98 (74%) were made by exome or genome sequencing. Of the diagnoses, 21% led to recommendations regarding changes in therapy, 37% led to changes in diagnostic testing, and 36% led to variant-specific genetic counseling. We defined 31 new syndromes. CONCLUSIONS: The UDN established a diagnosis in 132 of the 382 patients who had a complete evaluation, yielding a rate of diagnosis of 35%. (Funded by the National Institutes of Health Common Fund.)

BackgroundQuiescence (G0) is a transient, cell cycle-arrested state. By entering G0, cancer cells survive unfavorable conditions such as chemotherapy and cause relapse. While G0 cells have been studied at the transcriptome level, how post-transcriptional regulation contributes to their chemoresistance remains unknown.ResultsWe induced chemoresistant and quiescent (G0) leukemic cells by serum-starvation or chemotherapy treatment. To study post-transcriptional regulation in G0 leukemic cells, we systematically analyzed their transcriptome, translatome, and proteome. We find that our resistant G0 cells recapitulate gene expression profiles of in vivo chemoresistant leukemic and G0 models. In G0 cells, canonical translation initiation is inhibited; yet we find that inflammatory genes are highly translated, indicating alternative post-transcriptional regulation. Importantly, AU-rich elements (AREs) are significantly enriched in the up-regulated G0 translatome and transcriptome. Mechanistically, we find the stress-responsive p38 MAPK-MK2 signaling pathway stabilizes ARE mRNAs by phosphorylation and inactivation of mRNA decay factor, tristetraprolin (TTP) in G0. This permits expression of ARE-bearing TNFα and DUSP1 that promote chemoresistance. Conversely, inhibition of TTP phophorylation by p38 MAPK inhibitors and non-phosphorylatable TTP mutant decreases ARE mRNAs and sensitizes leukemic cells to chemotherapy. Furthermore, co-inhibiting p38 MAPK and TNFα—prior to or along with chemotherapy—substantially reduced chemoresistance in primary leukemic cells ex vivo and in vivo.ConclusionsThese studies uncover post-transcriptional regulation underlying chemoresistance in leukemia. Our data reveal the p38 MAPK-MK2-TTP axis as a key regulator of expression of ARE bearing mRNAs that promote chemoresistance. By disrupting this pathway, we developed an effective combination therapy against chemosurvival.

Interferon regulatory factor 2 binding protein-like (IRF2BPL) encodes a member of the IRF2BP family of transcriptional regulators. Currently the biological function of this gene is obscure, and the gene has not been associated with a Mendelian disease. Here we describe seven individuals who carry damaging heterozygous variants in IRF2BPL and are affected with neurological symptoms. Five individuals who carry IRF2BPL nonsense variants resulting in a premature stop codon display severe neurodevelopmental regression, hypotonia, progressive ataxia, seizures, and a lack of coordination. Two additional individuals, both with missense variants, display global developmental delay and seizures and a relatively milder phenotype than those with nonsense alleles. The IRF2BPL bioinformatics signature based on population genomics is consistent with a gene that is intolerant to variation. We show that the fruit-fly IRF2BPL ortholog, called pits (protein interacting with Ttk69 and Sin3A), is broadly detected, including in the nervous system. Complete loss of pits is lethal early in development, whereas partial knockdown with RNA interference in neurons leads to neurodegeneration, revealing a requirement for this gene in proper neuronal function and maintenance. The identified IRF2BPL nonsense variants behave as severe loss-of-function alleles in this model organism, and ectopic expression of the missense variants leads to a range of phenotypes. Taken together, our results show that IRF2BPL and pits are required in the nervous system in humans and flies, and their loss leads to a range of neurological phenotypes in both species.

Purpose Sixty-75% of individuals with rare and undiagnosed phenotypes remain undiagnosed after whole exome sequencing (ES). With standard ES reanalysis resolving 10–15% of the ES negatives, further approaches are necessary to maximize diagnoses in these individuals. Methods In 38 ES negative patients an individualized genomic-phenotypic approach was employed utilizing: A) Phenotyping; B) Reanalyses of FASTQ files, with innovative bioinformatics; C) Targeted molecular testing; D) Whole genome sequencing (WGS) and E) Conferring of clinical diagnoses when pathognomonic clinical findings occurred. Results Certain and Highly Likely diagnoses were made in 18/38 (47%) individuals, including identifying two new developmental disorders. The majority of diagnoses (>70%) were due to our bioinformatics, phenotyping and targeted testing identifying variants that were undetected or not prioritized on prior ES. WGS diagnosed 3/18 individuals, with structural variants not amenable to ES. Additionally, Tentative diagnoses were made in three (8%) and in five individuals (13%) candidate genes were identified. Overall, diagnoses/potential leads were identified in 26/38 (68%). Conclusions Our comprehensive approach to ES negatives maximizes the ES and clinical data for both diagnoses and candidate gene identification, without WGS in the majority. This iterative approach is cost-effective and is pertinent to the current conundrum of ES negatives.

Next-generation sequencing–based methods are being adopted broadly for genetic diagnostic testing, but the performance characteristics of these techniques with regard to test accuracy and reproducibility have not been fully defined. We developed a targeted enrichment and next-generation sequencing approach for genetic diagnostic testing of patients with inherited eye disorders, including inherited retinal degenerations, optic atrophy, and glaucoma. In preparation for providing this genetic eye disease (GEDi) test on a CLIA–certified basis, we performed experiments to measure the sensitivity, specificity, and reproducibility, as well as the clinical sensitivity, of the test. The GEDi test is highly reproducible and accurate, with sensitivity and specificity of 97.9 and 100%, respectively, for single-nucleotide variant detection. The sensitivity for variant detection was notably better than the 88.3% achieved by whole-exome sequencing using the same metrics, because of better coverage of targeted genes in the GEDi test as compared with a commercially available exome capture set. Prospective testing of 192 patients with inherited retinal degenerations indicated that the clinical sensitivity of the GEDi test is high, with a diagnostic rate of 51%. Based on quantified performance metrics, the data suggest that selective targeted enrichment is preferable to whole-exome sequencing for genetic diagnostic testing. Genet Med 17 4, 253–261.

Coenzyme Q10 (CoQ10) deficiency can manifest diversely, from isolated myopathy to multisystem involvement. Immune dysregulation has not been reported as a feature of the disease. We report a four-year old girl with failure to thrive, recurrent infections, developmental delay with hypotonia, and CoQ10 deficiency with impaired immune function, which improved after CoQ10 and immunoglobulin replacement therapy. Immune dysfunction in CoQ10 deficiency should be considered and treated appropriately.

Background The causes of intellectual disability (ID) are diverse and de novo mutations are increasingly recognised to account for a significant proportion of ID. Methods and results In this study, we performed whole exome sequencing on a large cohort of patients with ID or neurodevelopmental delay and identified four novel de novo predicted deleterious missense variants in HECW2 in six probands with ID/developmental delay and hypotonia. Other common features include seizures, strabismus, nystagmus, cortical visual impairment and dysmorphic facial features. HECW2 is an ubiquitin ligase that stabilises p73, a crucial mediator of neurodevelopment and neurogenesis. Conclusion This study implicates pathogenic genetic variants in HECW2 as potential causes of neurodevelopmental disorders in humans.

PurposeTo describe examples of missed pathogenic variants on whole-exome sequencing (WES) and the importance of deep phenotyping for further diagnostic testing.MethodsGuided by phenotypic information, three children with negative WES underwent targeted single-gene testing.ResultsIndividual 1 had a clinical diagnosis consistent with infantile systemic hyalinosis, although WES and a next-generation sequencing (NGS)-based ANTXR2 test were negative. Sanger sequencing of ANTXR2 revealed a homozygous single base pair insertion, previously missed by the WES variant caller software. Individual 2 had neurodevelopmental regression and cerebellar atrophy, with no diagnosis on WES. New clinical findings prompted Sanger sequencing and copy number testing of PLA2G6. A novel homozygous deletion of the noncoding exon 1 (not included in the WES capture kit) was detected, with extension into the promoter, confirming the clinical suspicion of infantile neuroaxonal dystrophy. Individual 3 had progressive ataxia, spasticity, and magnetic resonance image changes of vanishing white matter leukoencephalopathy. An NGS leukodystrophy gene panel and WES showed a heterozygous pathogenic variant in EIF2B5; no deletions/duplications were detected. Sanger sequencing of EIF2B5 showed a frameshift indel, probably missed owing to failure of alignment.ConclusionThese cases illustrate potential pitfalls of WES/NGS testing and the importance of phenotype-guided molecular testing in yielding diagnoses.

One major challenge encountered with interpreting human genetic variants is the limited understanding of the functional impact of genetic alterations on biological processes. Furthermore, there remains an unmet demand for an efficient survey of the wealth of information on human homologs in model organisms across numerous databases. To efficiently assess the large volume of publically available information, it is important to provide a concise summary of the most relevant information in a rapid user-friendly format. To this end, we created MARRVEL (model organism aggregated resources for rare variant exploration). MARRVEL is a publicly available website that integrates information from six human genetic databases and seven model organism databases. For any given variant or gene, MARRVEL displays information from OMIM, ExAC, ClinVar, Geno2MP, DGV, and DECIPHER. Importantly, it curates model organism-specific databases to concurrently display a concise summary regarding the human gene homologs in budding and fission yeast, worm, fly, fish, mouse, and rat on a single webpage. Experiment-based information on tissue expression, protein subcellular localization, biological process, and molecular function for the human gene and homologs in the seven model organisms are arranged into a concise output. Hence, rather than visiting multiple separate databases for variant and gene analysis, users can obtain important information by searching once through MARRVEL. Altogether, MARRVEL dramatically improves efficiency and accessibility to data collection and facilitates analysis of human genes and variants by cross-disciplinary integration of 18 million records available in public databases to facilitate clinical diagnosis and basic research.

Galloway-Mowat syndrome (GAMOS) is a severe autosomal-recessive disease characterized by the combination of early-onset steroid-resistant nephrotic syndrome (SRNS) and microcephaly with brain anomalies. To date, mutations of WDR73 are the only known monogenic cause of GAMOS and in most affected individuals the molecular diagnosis remains elusive. We here identify recessive mutations of OSGEP, TP53RK, TPRKB, or LAGE3, encoding the 4 subunits of the KEOPS complex in 33 individuals of 30 families with GAMOS. CRISPR/Cas9 knockout in zebrafish and mice recapitulates the human phenotype of microcephaly and results in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibits cell proliferation, which human mutations fail to rescue, and knockdown of either gene activates DNA damage response signaling and induces apoptosis. OSGEP and TP53RK molecularly interact and co-localize with the actin-regulating ARP2/3 complex. Furthermore, knockdown of OSGEP and TP53RK induces defects of the actin cytoskeleton and reduces migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identify 4 novel monogenic causes of GAMOS, describe the first link between KEOPS function and human disease, and delineate potential pathogenic mechanisms.

We report on a father and his two daughters diagnosed with Klippel–Feil syndrome (KFS) but with craniofacial differences (zygomatic and mandibular hypoplasia and cleft palate) and external ear abnormalities suggestive of Treacher Collins syndrome (TCS). The diagnosis of KFS was favored, given that the neck anomalies were the predominant manifestations, and that the diagnosis predated later recognition of the association between spinal segmentation abnormalities and TCS. Genetic heterogeneity and the rarity of large families with KFS have limited the ability to identify mutations by traditional methods. Whole exome sequencing identified a nonsynonymous mutation in POLR1D (subunit of RNA polymerase I and II): exon2:c.T332C:p.L111P. Mutations in POLR1D are present in about 5% of individuals diagnosed with TCS. We propose that this mutation is causal in this family, suggesting a pathogenetic link between KFS and TCS. © 2014 Wiley Periodicals, Inc.

Developing novel therapies that suppress self-renewal of leukemia stem cells may reduce the likelihood of relapses and extend long-term survival of patients with acute myelogenous leukemia (AML). AML1-ETO (AE) is an oncogene that plays an important role in inducing self-renewal of hematopoietic stem/progenitor cells (HSPCs), leading to the development of leukemia stem cells. Previously, using a zebrafish model of AE and a whole-organism chemical suppressor screen, we have discovered that AE induces specific hematopoietic phenotypes in embryonic zebrafish through a cyclooxygenase (COX)-2 and β-catenin-dependent pathway. Here, we show that AE also induces expression of the Cox-2 gene and activates β-catenin in mouse bone marrow cells. Inhibition of COX suppresses β-catenin activation and serial replating of AE(+) mouse HSPCs. Genetic knockdown of β-catenin also abrogates the clonogenic growth of AE(+) mouse HSPCs and human leukemia cells. In addition, treatment with nimesulide, a COX-2 selective inhibitor, dramatically suppresses xenograft tumor formation and inhibits in vivo progression of human leukemia cells. In summary, our data indicate an important role of a COX/β-catenin-dependent signaling pathway in tumor initiation, growth, and self-renewal, and in providing the rationale for testing potential benefits from common COX inhibitors as a part of AML treatments.

The presence of AML1-ETO (RUNX1-CBF2T1), a fusion oncoprotein resulting from a t(8;21) chromosomal translocation, has been implicated as a necessary but insufficient event in the development of a subset of acute myeloid leukemias (AML). While AML1-ETO prolongs survival and inhibits differentiation of hematopoietic stem cells (HSC), other contributory events are needed for cell proliferation and leukemogenesis. We have postulated that specific tumor suppressor genes keep the leukemic potential of AML1-ETO in check. In studying del(9q), one of the most common concomitant chromosomal abnormalities with t(8;21), we identified the loss of an apparent tumor suppressor, TLE4, that appears to cooperate with AML1-ETO to confer a leukemic phenotype. This study sought to identify the molecular basis of this cooperation. We show that the loss of TLE4 confers proliferative advantage to leukemic cells, simultaneous with an upregulation of a pro- inflammatory signature mediated through aberrant increases in Wnt signaling activity. We further demonstrate that inhibition of cyclooxygenase (COX) activity partly reverses the pro-leukemic phenotype due to TLE4 knockdown, pointing towards a novel therapeutic approach for myeloid leukemia.

Despite the recent acceleration in the discovery of genetic risk factors for intellectual disability (ID), the genetic etiology of ID is unknown in approximately half of cases and remains a major frontier of genetics in medicine and psychiatry. The distinction between syndromal and non-syndromal forms of ID is of great clinical importance, but the boundary between these clinical entities is difficult to ascertain for many genes of interest. ID is more common in men than in women, but the genetic explanation of this gender asymmetry is incompletely understood . This Review systematically examines the reported cases of X-linked ID caused by de novo loss-of-function mutations in the gene IQSEC2. This gene is largely known as a cause of X-linked non-syndromal ID in male patients. However, depending on the severity of the mutation, the phenotypic spectrum of IQSEC2-related ID can range from the classic X-linked non-syndromal form of the disease to a severe syndrome that has been reported in the context of de novo mutations only, in both male and female patients. Bioinformatic analysis suggests that truncation of the longer of the two protein isoforms of the gene can be sufficient to lead to the syndrome, which may be caused by the disruption of cell signaling and signal transduction pathways. The clinical features of the syndrome converge on a pattern of global developmental delay, deficits in social communication, stereotypical hand movements and hypotonia. In addition, many if not all of these patients have seizures, microcephaly and language regression in addition to delay. We argue that it is clinically appropriate to test for IQSEC2 mutations in male and female patients with this symptom profile but without a known genetic mutation.

Tle1 (transducin-like enhancer of split 1) is a corepressor that interacts with a variety of DNA-binding transcription factors and has been implicated in many cellular functions; however, physiological studies are limited. Tle1-deficient (Tle1(Δ/Δ)) mice, although grossly normal at birth, exhibit skin defects, lung hypoplasia, severe runting, poor body condition, and early mortality. Tle1(Δ/Δ) mice display a chronic inflammatory phenotype with increased expression of inflammatory cytokines and chemokines in the skin, lung, and intestine and increased circulatory IL-6 and G-CSF, along with a hematopoietic shift toward granulocyte macrophage progenitor and myeloid cells. Tle1(Δ/Δ) macrophages produce increased inflammatory cytokines in response to Toll-like receptor (TLR) agonists and lipopolysaccharides (LPS), and Tle1(Δ/Δ) mice display an enhanced inflammatory response to ear skin 12-O-tetradecanoylphorbol-13-acetate treatment. Loss of Tle1 not only results in increased phosphorylation and activation of proinflammatory NF-κB but also results in decreased Hes1 (hairy and enhancer of split-1), a negative regulator of inflammation in macrophages. Furthermore, Tle1(Δ/Δ) mice exhibit accelerated growth of B6-F10 melanoma xenografts. Our work provides the first in vivo evidence, to our knowledge, that TLE1 is a major counterregulator of inflammation with potential roles in a variety of inflammatory diseases and in cancer progression.

The era of personalized medicine has arrived, and with it a need for leaders in this discipline. This generation of trainees requires a cadre of new skill sets to lead the implementation of personalized medicine into mainstream healthcare. Traditional training programs no longer provide trainees with all the skills they will need to optimize implementation of this revolution now underway in medicine. Today’s trainees must manage clinical teams, act as clinical and molecular diagnostic consultants, train other healthcare professionals, teach future generations, and be knowledgeable about clinical trials to facilitate genomic-based therapies. To prepare trainees for the transition to junior faculty positions, contemporary genomic training programs must emphasize the development of these management, teaching, and clinical skills.

Item does not contain fulltext The ASXL genes (ASXL1, ASXL2, and ASXL3) participate in body patterning during embryogenesis and encode proteins involved in epigenetic regulation and assembly of transcription factors to specific genomic loci. Germline de novo truncating variants in ASXL1 and ASXL3 have been respectively implicated in causing Bohring-Opitz and Bainbridge-Ropers syndromes, which result in overlapping features of severe intellectual disability and dysmorphic features. ASXL2 has not yet been associated with a human Mendelian disorder. In this study, we performed whole-exome sequencing in six unrelated probands with developmental delay, macrocephaly, and dysmorphic features. All six had de novo truncating variants in ASXL2. A careful review enabled the recognition of a specific phenotype consisting of macrocephaly, prominent eyes, arched eyebrows, hypertelorism, a glabellar nevus flammeus, neonatal feeding difficulties, hypotonia, and developmental disabilities. Although overlapping features with Bohring-Opitz and Bainbridge-Ropers syndromes exist, features that distinguish the ASXL2-associated condition from ASXL1- and ASXL3-related disorders are macrocephaly, absence of growth retardation, and more variability in the degree of intellectual disabilities. We were also able to demonstrate with mRNA studies that these variants are likely to exert a dominant-negative effect, given that both alleles are expressed in blood and the mutated ASXL2 transcripts escape nonsense-mediated decay. In conclusion, de novo truncating variants in ASXL2 underlie a neurodevelopmental syndrome with a clinically recognizable phenotype. This report expands the germline disorders that are linked to the ASXL genes.

Quiescent (G0) cells are a clinically relevant fraction in several cancers, which include dormant cancer stem cells, and resist clinical therapy. G0 cells reveal extensive changes in gene expression at the protein and translation levels. We previously identified that the translation mechanism is altered in G0 cancer cells. MicroRNAs, noncoding RNAs that target distinct mRNAs to alter gene expression—were found to associate with an important RNA-binding protein, and enable specialized functions in G0—where they recruit non-canonical translation factors to regulate specific mRNA translation. We find that G0 leukemic cells show similar proteome and translatome to cells isolated post-chemotherapy. These data suggest that specialized post-transcriptional and translational mechanisms in G0 leukemic cells regulate a distinct translatome to mediate chemoresistance. To understand the role of post-transcriptional and translational regulation in chemoresistance, we compared global RNA, translational and proteome profiling in chemoresistant G0 acute monocytic leukemic (AML) cells. We find that chemotherapy or G0 induction leads to DNA damage responsive ATM and stress signaling, which alter post-transcriptional and translational mechanisms. ATM and stress activated p38 MAPK/MK2 increase AU-rich-element (ARE) bearing pro-inflammatory cytokine and immune gene mRNAs by regulating a key ARE RNA binding protein, and by activating STAT1/interferon pathway to alter canonical translation. AREs are present on 3'UTRs of critical, tightly regulated oncogenes and cytokines to post-transcriptionally control their expression. These changes permit translation of ARE bearing pro-inflammatory cytokine TNFα, and immune and cell-migration modulators that promote survival. Co-inhibiting p38 MAPK and TNFα that promote anti-apoptosis—prior to or alongwith chemotherapy—decreases chemoresistance in AML cell lines, in vivo, and in patient samples, without affecting normal cells. These studies reveal a pro-inflammatory subpopulation in AML that mediates chemoresistance, enabled by DNA damage- and stress-regulated post-transcriptional and translational mechanisms that are mediated by AU-rich-elements and a critical ARE RNA binding protein. Disrupting ARE regulation reduces TNFα and chemoresistance, revealing AREs and an important ARE RNA binding protein as key regulators of inflammation-mediated chemoresistance. These studies reveal the significance of post-transcriptional regulation of inflammation/immune gene-mediated chemoresistance. Correspondence: vasudevan.shobha@mgh.harvard.edu Citation Format: Sooncheol Lee, Samuel S. Truesdell, Syed I. Bukhari, Myriam Boukhali, Dongjun Lee, Maria A. Mazzola, Radhika Raheja, Adam Langenbucher, Nicholas J. Haradhvala, Michael Lawrence, Roopali Gandhi, David A. Sweetser, Wilhelm Haas, Shobha Vasudevan. A post-transcriptional program of chemoresistance regulators in quiescent cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4443.

Next-generation sequencing has evolved technically and economically into the method of choice for interrogating the genome in cancer and inherited disorders. The introduction of procedural code sets for whole-exome and genome sequencing is a milestone toward financially sustainable clinical implementation; however, achieving reimbursement is currently a major challenge. As part of a prospective quality-improvement initiative to implement the new code sets, we adopted Agile, a development methodology originally devised in software development. We implemented eight functionally distinct modules (request review, cost estimation, preauthorization, accessioning, prebilling, testing, reporting, and reimbursement consultation) and obtained feedback via an anonymous survey. We managed 50 clinical requests (January to June 2015). The fraction of pursued-to-requested cases (n = 15/50; utilization management fraction, 0.3) aimed for a high rate of preauthorizations. In 13 of 15 patients the insurance plan required preauthorization, which we obtained in 70% and ultimately achieved reimbursement in 50%. Interoperability enabled assessment of 12 different combinations of modules that underline the importance of an adaptive workflow and policy tailoring to achieve higher yields of reimbursement. The survey confirmed a positive attitude toward self-organizing teams. We acknowledge the individuals and their interactions and termed the infrastructure: human pipeline. Nontechnical barriers currently are limiting the scope and availability of clinical genomic sequencing. The presented human pipeline is one approach toward long-term financial sustainability of clinical genomics.

Mutations in mitochondrial aminoacyl-tRNA synthetases are an increasingly recognized cause of human diseases, often arising in individuals with compound heterozygous mutations and presenting with system-specific phenotypes, frequently neurologic. FARS2 encodes mitochondrial phenylalanyl transfer ribonucleic acid (RNA) synthetase (mtPheRS), perturbations of which have been reported in 6 cases of an infantile, lethal disease with refractory epilepsy and progressive myoclonus. Here the authors report the case of juvenile onset refractory epilepsy and progressive myoclonus with compound heterozygous FARS2 mutations. The authors describe the clinical course over 6 years of care at their institution and diagnostic studies including electroencephalogram (EEG), brain magnetic resonance imaging (MRI), serum and cerebrospinal fluid analyses, skeletal muscle biopsy histology, and autopsy gross and histologic findings, which include features shared with Alpers-Huttenlocher syndrome, Leigh syndrome, and a previously published case of FARS2 mutation associated infantile onset disease. The authors also present structure-guided analysis of the relevant mutations based on published mitochondrial phenylalanyl transfer RNA synthetase and related protein crystal structures as well as biochemical analysis of the corresponding recombinant mutant proteins.

Acute myeloid leukemia (AML) is a clonal disease characterized by heterogeneous involvement of hematopoietic stem cell/progenitor cell populations. Using FLT3 internal tandem duplication (FLT3/ITD) as a molecular marker, we tested the hypothesis that clinical outcome in AML correlates with disease involvement of CD34(+)/CD33(-) precursors. Diagnostic specimens from 24 children with FLT3/ITD-positive AML were sorted by fluorescence-activated cell sorting (FACS), and resultant CD34(+)/CD33(-) and CD34(+)/CD33(+) progenitors were analyzed directly and after colony-forming cell (CFC) assay for the presence of FLT3/ITD. FLT3/ITD was present in all CD34(+)/CD33(+) patient samples. In contrast, FLT3/ITD was detected in CD34(+)/CD33(-) progenitors in only 19 of 24 samples. A bipotent progenitor was affected in a subset of patients, as evidenced by the presence of FLT3/ITD in both granulocyte-macrophage colony-forming unit (CFU-GM) and erythroid burst-forming unit (BFU-E) colonies. Those patients in whom CD34(+)/CD33(-) precursors harbored the FLT3/ITD had worse clinical outcome; actuarial event-free survival (EFS) at 4 years from study entry for those patients with and without FLT3/ITD detection in CD34(+)/CD33(-) progenitors was 11% +/- 14% versus 100% +/- 0%, respectively (P = .002). This study suggests that FLT3/ITD involvement in CD34(+)/CD33(-) precursors is heterogeneous and that detection of the mutation in the less-mature progenitor population may be associated with disease resistance.

Deletion of the long arm of chromosome 9, del(9q), is a recurring chromosomal aberration in acute myeloid leukemia (AML) that is frequently associated with t(8;21). The critical gene products affected by del(9q) are unknown but likely cooperate with the AML1/ETO fusion gene created by t(8;21) in leukemogenesis. In 43 AML samples with del(9q), we used high-density microsatellite markers to define the commonly deleted region (CDR) to less than 2.4 Mb. We found no homozygous loss at any locus tested. The CDR contains 7 known genes, FRMD3, UBQLN1, GKAP42, KIF27, HNRPK, SLC28A3, and NTRK2, and 4 novel genes, RASEF, C9orf103, C9orf64, and C9orf76. In addition, TLE1 and TLE4 are adjacent to the CDR. We performed a comprehensive mutational analysis of the coding regions of all these genes. No sequence variations absent in normal controls were seen in more than a single del(9q) AML sample. Expression of 7 of the 10 genes examined was significantly down-regulated in del(19q)AML as compared with the CD34-purified progenitors from normal individuals, a pattern distinct from that seen in AML samples with a normal karyotype. The results of our studies are consistent with a model of tumor suppression mediated by haploinsufficiency of critical genes in del(9q) AML. © 2005 Wiley-Liss, Inc.

Activating mutations of receptor tyrosine kinases (RTKs) and their downstream affectors are common in acute myeloid leukemia (AML). We performed mutational analysis of FLT3, c-kit, c-fms, vascular endothelial growth factor (VEGF) receptors (Flt-1, KDR [kinase domain receptor]), and ras genes in a group of 91 pediatric patients with AML treated on Children's Cancer Group clinical trial CCG-2891. Forty-six percent of patients had activating mutations of FLT3 (24.5%), c-kit (3%), or ras (21%) genes. Mutation-positive patients had a higher median diagnostic white blood cell (WBC) count (71.5 vs 19.6 × 109/L; P = .005) and lower complete remission rate (55% versus 76%; P = .046) than mutation-negative patients. The Kaplan-Meier estimate of overall survival (OS) for patients with and without an activating mutation was 34% versus 57%, respectively (P = .035). However, within this group, patients with FLT3/ALM (activation loop mutation) had good outcomes (OS, 86%). Exclusion of the FLT3/ALM from analysis decreased the OS for the remaining mutation-positive patients to 26% (P = .003). Ten of the 23 mutation-positive and 11 of the 34 mutation-negative patients received an allogeneic bone marrow transplant (BMT) in first complete remission (CR). In the mutation-positive group, the disease-free survival (DFS) for the allogeneic BMT recipients was 72% versus 23% for the 13 patients who received chemotherapy or autologous BMT (P = .01). DFS for the mutation-free patients with and without allogeneic BM transplantation was 55% and 40%, respectively (P = .38). Activating mutations in the RTK/ras signaling pathway are common in pediatric AML, and their presence may identify a population at higher risk of poor outcome who may benefit from allogeneic BM transplantation.

Mitochondrial disorders are a heterogeneous group of multisystem disorders linked to dysfunction of the mitochondrion, an organelle critical to cellular metabolism, diagnosed via a multifaceted approach that involves clinical assessment, family history, and genetic and biochemical testing. Systems assessed in existing clinical criteria for mitochondrial disorders include the central nervous, neuromuscular, cardiovascular, hematologic, gastrointestinal, endocrine, renal, ophthalmologic, auditory, hepatic, and dermatologic systems. To our knowledge, no current clinical criteria consider infections or immunologic dysfunction as components of primary mitochondrial disorders despite scientific literature that links mitochondria and the immune system. The current study was undertaken to assess the occurrence of infection and immunodysfunction among patients with mitochondrial disorders. From the Massachusetts General Hospital mitochondrial disorders database of >250 patients, we selected those with an established molecular etiology or a clinical “probable” or “definite” diagnosis of mitochondrial disorder (see Table E1 in this article’s Online Repository at www.jaciinpractice.org) and further supported by results from biopsy, electron transport chain enzymatic abnormalities, and/or molecular diagnostics (targeted sequencing based on clinical presentation, or broader screening, primarily “MitoExome” analysis). The majority of electron transport chain enzyme function testing to assess enzyme activity

A genome-wide screening for loss of heterozygosity (LOH), a marker for possible involvement of tumor suppressor genes, was conducted in 53 children with de novo acute myelogenous leukemia (AML). A total of 177 highly polymorphic microsatellite repeat markers were used in locus-specific polymerase chain reactions. This comprehensive allelotyping employed flow-sorted cells from diagnostic samples and whole-genome amplification of DNA from small, highly purified samples. Nineteen regions of allelic loss in 17 patients (32%) were detected on chromosome arms 1q, 3q, 5q, 7q (n = 2), 9q (n = 4), 11p (n = 2), 12p (n = 3), 13q (n = 2), 16q, 19q, and Y. The study revealed a degree of allelic loss underestimated by routine cytogenetic analysis, which failed to detect 9 of these LOH events. There was no evidence of LOH by intragenic markers for p53, Nf1, or CBFA2/AML1. Most lymphocytes lacked the deletions, which were detected only in the leukemic myeloid blast population. Analysis of patients' clinical and biologic characteristics indicated that the presence of LOH was associated with a white blood cell count of 20 x 10(9)/L or higher but was not correlated with a shorter overall survival. The relatively low rate of LOH observed in this study compared with findings in solid tumors and in pediatric acute lymphoblastic leukemia and adult AML suggests that tumor suppressor genes are either infrequently involved in the development of pediatric de novo AML or are inactivated by such means as methylation and point mutations. Additional study is needed to determine whether these regions of LOH harbor tumor suppressor genes and whether specific regions of LOH correlate with clinical characteristics. (Blood. 2001;98:1188-1194)

The Flt3 gene encodes a tyrosine kinase receptor that regulates proliferation and differentiation of hematopoietic stem cells. An internal tandem duplication of the Flt3 gene (Flt3/ITD) has been reported in acute myelogenous leukemia (AML) and may be associated with poor prognosis. We analyzed diagnostic bone marrow specimens from 91 pediatric patients with AML treated on Children's Cancer Group (CCG)-2891 for the presence of the Flt3/ITD and correlated its presence with clinical outcome. Fifteen of 91 samples (16.5%) were positive for the Flt3/ITD. Flt3/ITD-positive patients had a median diagnostic white count of 73 800 compared with 28 400 for the Flt3/ITD-negative patients (P = .05). The size of the duplication ranged from 21 to 174 base pairs (bp). Nucleotide sequencing of the abnormal polymerase chain reaction products demonstrated that all duplications involved exon 11 of theFlt3 gene and also preserved the reading frame. Lineage restriction analysis revealed that Flt3/ITD was not present in the lymphocytes, suggesting a lack of stem cell involvement for this mutation. None of the Flt3/ITD-positive patients had unfavorable cytogenetic markers, and there was no predominance of a particular FAB class. The remission induction rate was 40% in Flt3/ITD-positive patients compared with 74% in Flt3/ITD-negative ones (P = .005). The Kaplan-Meier estimates of event-free survival at 8 years for patients with and without Flt3/ITD were 7% and 44%, respectively (P = .002). Multivariate analysis demonstrated that presence of the Flt3/ITD was the single most significant, independent prognostic factor for poor outcome (P = .009) in pediatric AML.

Complete colonization of the gut by enteric neural precursors depends on activation of ednrB and Ret receptors by their respective ligands, edn3 and gdnf. Mutations that eliminate expression of either ligand or either receptor produce intestinal aganglionosis in rodents and humans. Embryos homozygous for the lethal spotted (ls) allele, a loss of function mutation in the edn3 gene, have no ganglion cells in their terminal large intestines and are spotted, due to incomplete colonization of the skin by melanocyte precursors. Expression of edn3 in enteric neural precursors of transgenic mice compensates fully for deficient endogenous edn3 in ls/ls embryos. The effects of the edn3 transgene are dose-dependent, as lower levels of expression in one line prevent aganglionosis in only a subset of animals and reduce, but fail to eliminate, piebaldism. In contrast, expression of neither constitutively active Ret nor activated ras in enteric neural progenitors alters the severity of aganglionosis or piebaldism in ls/ls mice. Given the spatial and temporal pattern of edn3-transgene expression, our results suggest that edn3/ednrB signals are not required prior to the arrival of crest cells in the gut and endrB stimulation elicits distinct cellular responses from Ret or ras activation. Dev Dyn 2000;217:120–132. © 2000 Wiley-Liss, Inc.

Sympathetic neurons, enteric neurons and adrenal chromaffin cells all derive from the neural crest. During development these cells migrate, proliferate, survive and differentiate in a highly controlled fashion influenced by local signals encountered during their migration. Aberrations of these processes are responsible for a variety of developmental defects and malignancies. Many of the environmental signals influencing these precursor cells activate receptor tyrosine kinases that can signal, at least in part, via Ras pathways. To assess the extent to which Ras can alter neuroblast cell number and fate in vivo, we expressed activated H-Ras in transgenic mice using the dopamine-beta-hydroxylase promoter, which directs expression to these cells prior to and after their differentiation. Ganglioneuromas and occasional neuroblastomas formed in the adrenal gland and preaortic sympathetic ganglia. Curiously, neurons of the superior cervical ganglia and the gut were largely unaffected despite demonstrated expression of activated Ras. The sensitivity of preaortic sympathetic neurons and adrenal chromaffin cells to the effects of oncogenes such as Ras may explain the predilection of neuroblastomas in humans to these sites. The ability to analyse neuroblastoma development in these mice may shed light on the molecular basis of certain types of human neuroblastoma.

Newborn screening (NBS) using tandem mass spectrometry (MS/MS) permits detection of neonates with Glutaric Aciduria-Type II (GA-II). We report follow-up of positive GA-II screens by the New England Newborn Screening Program.1.5 million infants were screened for GA-II (Feb 1999-Dec 2012). Specialist consult was suggested for infants with two or more acylcarnitine elevations suggestive of GA-II.82 neonates screened positive for GA-II, 21 weighing1.5 kg and 61 weighing ≤ 1.5 kg. Seven (one weighing1.5 kg), were confirmed with GA-II. Four of these had the severe form (died1 week). The other three have a milder form and were identified because of newborn screening. Two (ages5 years) have a G-Tube in place, had multiple hospitalizations and are slightly hypotonic. The third infant remains asymptomatic (9 months old). Two GA-II carriers were also identified. The remaining positive screens were classified as false positives (FP). Six infants (1.5 kg) classified as FP had limited diagnostic work-up. Characteristics and outcomes of all specimens and neonates with a positive screen were reviewed, and marker profiles of the cases and FP were compared to identify characteristic profiles.In addition to the severe form of GA-II, milder forms of GA-II and some GA-II carriers are identified by newborn screening. Some positive screens classified as FP may be affected with a milder form of the disorder. Characteristic GA-II profiles, quantified as GA-II indexes, may be utilized to predict probability of disorder and direct urgency of intervention for positive screens.

Mice homozygous for the piebald lethal (sl) mutation, which have a complete deletion of endothelin receptor-B, fail to form ganglion cells in the distal large intestine and are nearly devoid of cutaneous melanocytes. These phenotypic features stem from incomplete colonization of the hindgut and skin by neural crest-derived neuroblasts and melanoblasts, respectively. We have used expression of a transgene, dopamine-beta-hydroxylase-nlacZ, to study colonization of the enteric nervous system in sl/sl embryos and sl/sl wild-type chimeric mice. Enteric neuroblasts derived from the vagal neural crest colonize the developing foregut, midgut and distal small intestine of sl/sl embryos in a cranial-to-caudal manner indistinguishable from sl/+ or +/+ embryos. However, colonization of the large intestine is retarded and the distal large intestine is never colonized, a developmental defect identical to that observed in lethal spotted (endothelin-3 deficient) embryos. The coat pigmentation and relative distributions of mutant and wild-type ganglion cells in sl/sl wild-type chimeras indicate that the defect associated with endothelin receptor-B gene deletion is not strictly neuroblast autonomous (independent of environmental factors). Instead, intercellular interactions downstream of the endothelin receptor-B mediate complete colonization of the skin and gut by neural crest cells.

Acute myeloid leukemia (AML) accounts for 15–20 % of the acute leukemias in children and 80 % of acute leukemia in adults [1, 2]. Significant advances as the result of multigroup cooperative trials have transformed this once uniformly fatal disease into one with a cure rate in children approaching 60 % [3], although the results are still significantly worse in adults. The difference in clinical outcome between childhood and adult AML is in part reflected by differences in the underlying biology. Recent advances in our understanding of stem cell physiology and the molecular basis of AML have elucidated the heterogeneity of AML and are leading to the identification of novel targets for therapy [4, 5]. The improvement in overall survival for children and young adults with AML can be attributed to better supportive care and advances in the application of chemotherapy and bone marrow transplantation. Future advancements in the treatment of children with AML will likely come about from the use of targeted therapies in conjunction with current chemotherapy regimens.

Associated with approximately 8-13% cases of acute myeloid leukemia (AML), t (8;21) results in the formation of the fusion oncoprotein AML1-ETO (AE), which appears to promote hematopoietic precursor expansion through aberrant regulation of canonical AML1 targets. Multiple lines of evidence indicate AE is insufficient for leukemogenesis and requires cooperative secondary mutations. Interstitial deletions of chromosome 9 are one of the more frequent associated cytogenetic abnormalities seen with t(8;21). We previously identified that knockdown of TLE1 and TLE4, members of the Groucho/TLE family of co-repressors residing in a commonly deleted region of chromosome 9 in t (8;21) del (9q) AML cooperates with AE to drive myeloblastic proliferation and cause a leukemic phenotype. In this study, we show how TLE4 loss in the context of AML1-ETO expression results in cell proliferation, decreased apoptosis, blocked differentiation, and increased resistance to chemotherapy and that these effects are mediated through an upregulation of cyclooxygenase (COX)-dependent inflammation. TLE4 knockdown via shRNA (shTLE4) in Kasumi-1 cells (derived from a patient with t (8;21) AML) resulted in increased cell cycling and decreased cell death that is concomitant with significantly lower levels of p16Ink4 and p27Kip1 expression, both major regulators of the G1/S cell cycle checkpoint. RNAseq with GSEA analysis identified enrichment of pathways related to IFN signaling and inflammation within upregulated genes in shTLE4 cells compared to control. Filtering for genes with log2 fold change greater than 0.6, we identified three genes related to inflammation (PTGER4, CEBPb, FOS). As demonstration of the relevance of these findings, we showed these expression levels were also higher in primary human del (9q) and t (8;21) del (9q) leukemia samples compared to normal CD34+ cells; revealing a potential leukemogenic role for inflammatory pathways in del (9q) and t (8;21) del (9q) AML. Using a sub-lethal dose of indomethacin (50uM INDM), we found modulating the COX-dependent inflammatory axis decreased growth of shTLE4 Kasumi-1 cells, decreased cell cycling, and increased dead populations. Concomitantly, INDM treatment was able to reverse shTLE4-induced increases in PTGER4, CEBPb, FOS, and IL1b expression to levels similar to those in control cells. Given the ability to induce myeloblastic proliferation with TLE knockdown and AE expression in zebrafish, we investigated whether shTLE4 causes changes in key myeloid differentiation regulators in Kasumi-1 cells. qPCR analysis demonstrated significantly repressed ELANE, MPO, and PU.1 expression in shTLE4-treated cells, which is relieved with the addition of INDM. TLE4 knockdown in HL60 cells (derived from t (15;17) PML/RARA leukemia) increased levels of FOS, IL1b, CEBPb, and PTGER4. At the same time, shTLE4 significantly reduced CD11b+ and CD11b+ CD14+ populations while decreasing expression of PU.1 and MYB - demonstrating a similar inflammatory gene signature associated with a myeloid differentiation block found in the Kasumi-1 model. We next wanted to examine whether inhibiting COX-dependent inflammation can be synergistic with existing therapies. INDM significantly increased the cell death and apoptosis seen in both control cells as well as cells with TLE4 knockdown treated with AraC. The loss of TLE4 conferred resistance to combination treatment with indomethacin and AraC as evidenced by increased cell proliferation and decreased cell death. However, we observed that cells with TLE4 knockdown were significantly more sensitive to the combination of sunitinib and INDM, pointing to a potentially more therapeutic and less toxic treatment regimen for this otherwise resistant cell population. In this study, we have demonstrated that loss of TLE4 appears to contribute to leukemogenesis through differentiation arrest and increased proliferation mediated through upregulation of COX-dependent inflammatory pathways. Our findings suggest that COX inhibition may serve as an effective adjuvant therapy for AML. Disclosures No relevant conflicts of interest to declare.

Retinoblastoma and Wilms’ tumor represent two childhood tumors with both sporadic and familial forms. Delineation of the molecular etiology of these cancers identified the retinoblastoma gene Rb as the first example of a tumor suppressor gene and provided the first example of imprinting and cancer predisposition in Wilms’ tumor. In this chapter, we review the clinical features and genetics of these two disorders and the implications for management, early detection and potential prevention of these disorders.

It has been proposed that inhibitors of an oncogene's effects on multipotent hematopoietic progenitor cell differentiation may change the properties of the leukemic stem cells and complement the clinical use of cytotoxic drugs. Using zebrafish, we developed a robust in vivo hematopoietic differentiation assay that reflects the activity of the oncogene AML1-ETO. Screening for modifiers of AML1-ETO-mediated hematopoietic dysregulation uncovered unexpected roles of COX-2- and beta-catenin-dependent pathways in AML1-ETO function. This approach may open doors for developing therapeutics targeting oncogene function within leukemic stem cells.

Deletions on chromosome 9q are seen in a subset of acute myeloid leukemia (AML) cases and are specifically associated with t(8;21) AML. We previously defined the commonly deleted region in del(9q) AML and characterized the genes in this interval. To determine the critical lost gene(s) that might cooperate with the AML1-ETO fusion gene produced by t(8;21), we developed a set of shRNAs directed against each gene in this region. Within this library, shRNAs to TLE1 and TLE4 were the only shRNAs capable of rescuing AML1-ETO expressing U937T-A/E cells from AML1-ETO–induced cell-cycle arrest and apoptosis. Knockdown of TLE1 or TLE4 levels increased the rate of cell division of the AML1-ETO–expressing Kasumi-1 cell line, whereas forced expression of either TLE1 or TLE4 caused apoptosis and cell death. Knockdown of Gro3, a TLE homolog in zebrafish, cooperated with AML1-ETO to cause an accumulation of noncirculating hematopoietic blast cells. Our data are consistent with a model in which haploinsufficiency of these TLEs overcomes the negative survival and antiproliferative effects of AML1-ETO on myeloid progenitors, allowing preleukemic stem cells to expand into AML. This study is the first to implicate the TLEs as potential tumor suppressor genes in myeloid leukemia.

The tyrosine kinase receptor Ret is expressed in the ureteric bud and is required for normal renal development. Constitutive loss of Ret, its co-receptor gfralpha-1, or the ligand glial cell line-derived neurotrophic factor results in renal agenesis. Transgenic embryos that express a constitutively active form of Ret (Ret(MEN2B)) under the control of the dopamine-beta-hydroxylase (DbetaH) promoter develop profound neuroglial hyperplasia of their sympathetic ganglia and adrenal medullae. Embryos from two independent DbetaH-Ret(MEN2B)-transgenic lines exhibit renal malformations. In contrast with ret-/- embryos, renal maldevelopment in DbetaH-Ret(MEN2B)-transgenic embryos results from primary changes in sympathoadrenal organs extrinsic to the kidney. The ureteric bud invades the metanephric mesenchyme normally, but subsequent bud branching and nephrogenesis are retarded, resulting in severe renal hypoplasia. Ablation of sympathoadrenal precursors restores normal renal growth in vivo and in vitro. We postulate that disruption of renal development results because Ret(MEN2B) derived from the hyperplastic nervous tissue competes with endogenous renal Ret for gfralpha-1 or other signaling components. This hypothesis is supported by the observation that renal malformations, which do not normally occur in a transgenic line with low levels of DbetaH-Ret(MEN2B) expression, arise in a gdnf+/- background. However, renal maldevelopment was not recapitulated in kidneys that were co-cultured with explanted transgenic ganglia in vitro. Our observations illustrate a novel pathogenic mechanism for renal dysgenesis that may explain how putative activating mutations of the RET gene can produce a phenotype usually associated with RET deficiency.

Multiple endocrine neoplasia type 2B (MEN2B) is an autosomal dominant syndrome characterized by the development of medullary thyroid carcinoma, pheochromocytomas, musculoskeletal anomalies and mucosal ganglioneuromas. MEN2B is caused by a specific mutation (Met918--Thr) in the RET receptor tyrosine kinase. Different mutations of RET lead to other conditions including MEN2A, familial medullary thyroid carcinoma and intestinal aganglionosis (Hirschsprung disease). Transgenic mice were created using the dopamine beta-hydroxylase promoter to direct expression of RET(MEN2B) in the developing sympathetic and enteric nervous systems and the adrenal medulla. DbetaH-RET(MEN2B) transgenic mice developed benign neuroglial tumors, histologically identical to human ganglioneuromas, in their sympathetic nervous systems and adrenal glands. The enteric nervous system was not affected. The neoplasms in DbetaH-RET(MEN2B) mice were similar to benign neuroglial tumors induced in transgenic mice by activated Ras expression under control of the same promoter. Levels of phosphorylated MAP kinase were not increased in the RET(MEN2B)-induced neurolglial proliferations, suggesting that alternative pathways may play a role in the pathogenesis of these lesions. Transgenic mice with the highest levels of DbetaH-RET(MEN2B) expression, unexpectedly developed renal malformations analogous to those reported with loss of function mutations in the Ret gene.

Dominant megacolon (Dom) is a mutation in an uncharacterized murine gene which is associated with intestinal aganglionosis and the focal absence of melanocytes in heterozygous animals. The phenotype of Dom/+ heterozygotes is similar to the lethal spotted and piebald lethal mutations, which are due to defects in endothelin-mediated intercellular signals. In this study, the DbetaH-nlacZ transgenic marker for enteric neural crest cells is used to study the distribution of enteric neurons and their precursors in Dom/+ mice and embryos. Vagal neural crest-derived cells in wild-type embryos colonize the gut in a cranial-to-caudal progression. In Dom/+ embryos, colonization was retarded from the earliest stages examined (embryonic Day 11.0), including progression through the small intestine. The early onset of this defect contrasts with impaired neural crest colonization associated with the lethal spotted and piebald lethal mutations which manifest only in the large intestine. Analysis of Dom/+ - +/+ aggregation chimeras indicated that defective colonization is not an autonomous (intrinsic) property of Dom/+ neuroblasts, but like lethal spotted and piebald lethal, the Dominant megacolon mutation directly or indirectly affects microenvironmental signals which influence the migration, proliferation, and/or survival of enteric neural crest cells.

A 16-year-old boy had intermittent chorea, delirium, and vertical gaze palsy precipitated by febrile illness. Nonketotic hyperglycinemia was confirmed by measurement of liver and lymphoblast glycine cleavage enzyme activity. Deficient but residual enzyme activity was demonstrated in both tissues, possibly accounting for the mild phenotype. Confirmation of an atypical variant of nonketotic hyperglycinemia with residual glycine cleavage enzyme activity has important implications for diagnosis and treatment.

The mouse receptor tyrosine kinase (RTK) NEP, also called Ptk-3, is widely expressed, with high levels in proliferating neuroepithelia of mouse embryos. The recently described human discoidin domain receptor (DDR) has a predicted amino acid sequence 93% identical to that of murine NEP and may be its human homologue. We have mapped the gene encoding NEP in human and mouse by fluorescence in situ hybridization using a mouse cDNA probe. The NEP/Nep gene maps to human chromosome 6p21.3 and mouse chromosome 17C, respectively. This places the NEP/Nep gene at, or near, the major histocompatibility (MHC) locus—HLA in human and H2 in mouse, respectively. Based on its pattern of expression during development, NEP and Nep represent candidate genes for several MHC-linked developmental abnormalities in human and mouse.

Abstract 1202 Transducin-like/Enhancer of Split 4 (TLE4), a homolog of the master developmental regulator in Drosophila, Groucho, is a co repressor previously described by our group as the putative tumor suppressor lost in a subset of t(8:21) AML patients also harboring 9q deletion. TLE4 has been found to be of fundamental importance in the repressive function of multiple highly conserved transcriptional complexes, including Hes1, Tcf/Lef, CSL, Runx1-3, and Pax5, all of which have been shown to be intimately connected to normal steady state hematopoiesis and deregulated in malignancy. Owing to its deletion in leukemia and its ability to confer functionality to a variety of signaling networks, our lab sought to investigate its role in hematopoiesis by generating a murine knockout model for Tle4. While normal at birth, within 2–3 weeks of age knockout (KO) animals become runted and eventually moribund by the time of weaning. Gross examination of the spleen and thymus in 3 week old animals reveals significant atrophy of these organs with severe abnormalities in normal tissue architecture. Tle4−/− mice lack clear demarcations between medullary and cortical regions of the thymus, in addition to having a significant increase in apoptosis seen via TUNEL staining. FACS analysis of the thymus shows CD4/CD8 double positive cells are heavily reduced in KOs, with an increase in both single positive mature populations. Additionally, there is a partial, but not complete, block in T cell progenitor development through TCR rearrangement (DN2-DN3 stages). Structural defects are seen in the spleen as well, with a failure in the formation of follicular zones. B cells are significantly reduced with an increase in CD11b+ cells in the spleen, in addition to a shift in the percentages of B1, B2, and marginal zone B cells in some animals. Histological examination of the long bones reveals dramatic abnormalities, both in hematopoietic and bone lineages. Tle4−/− mice exhibit a reduction in calcified bone, a dysplastic and reduced hypertrophic zone, and reabsorption of the trabecular region. These mice also have a gradual clearing of the marrow itself, with 2 week old animals first showing evidence of cytopenia that then progresses to full marrow failure by 3–3.5 weeks of age. The bone marrow exhibits a marked leukocytopenia, with a clear B lymphocytopenia as in the spleen. Furthermore, within the bone marrow, there is an increase in the number of CD4 and decrease in the number of CD8 T cells. ProB staging of developing B cells also trends towards a partial arrest in the transitions from the A through C fractions. When analyzed for progenitor populations, KO mice show severe reductions in the number of common lymphoid progenitors, while myeloid progenitors show no particular shifts in population frequency. Finally, there is a significant decrease in HSCs, defined as lin-Sca1+Ckit++ (LSK). In order to assess whether the hematological phenotype observed in these mice are of a cell intrinsic or extrinsic nature, transplants were performed using embryonic day 13.5 fetal liver and 2 week old bone marrow. Analysis of recipient animals 16 weeks after transplant revealed leukocytopenia with a specific reduction in B cells in the blood, however the T cell and LSK defects seen in donor animals was not observed. Furthermore, when LSK CD34+ Flt3+ or – cells from KO or WT 2 week old animals were plated on OP9 cells over-expressing Deltex1 or GFP, there were no defects in T, B, or CD11b+ cell production. The unique spectrum of phenotypic consequences concomitant to Tle4 deletion argues for a complex yet fundamental role in the maintenance of the hematopoietic niche. As illustrated by the ability to recapitulate some yet not all of the KO hematological aberrations in transplants, it appears that Tle4's involvement in lineage commitment is heterotypic in nature, an unsurprising finding considering its direct involvement in multiple highly conserved signaling pathways. Considering its principal role in those pathways, continued efforts to investigate the role the TLE family of corepressors play in the bone marrow niche may prove vital in reworking our paradigms for both normal and malignant hematopoiesis. Figure 1: Tle4 deletion causes alterations in LSK and common lymphoid progenitor frequencies. (Controls n=7, KO n=8; *p Figure 2: Representative Flow plots illustrating progenitor and LSK defects in Tle4−/− mice. Figure 2:. Representative Flow plots illustrating progenitor and LSK defects in Tle4−/− mice. Disclosures: No relevant conflicts of interest to declare.

Abstract 1197 TLE1 belongs to the Groucho/TLE family of co-repressors that act as master regulators during development affecting segmentation, neurogenesis, myogenesis, and multiple cell fate decisions. TLE1 modulate several major signaling pathways including Wnt and Notch, and specifically interacts with multiple transcription factors involved in hematopoiesis such TCF/LEF, HES1, RUNX1/AML. TLE1 has also been implicated in Crohn's disease via its interaction with NOD2, a regulator of NFkB. Our laboratory identified TLE1 as a likely AML tumor suppressor gene, commonly deleted in subgroups of AML, and others have shown its role as a tumor suppressor gene in myeloid and other hematopoietic malignancies. To better understand the role of TLE1 in hematopoiesis and leukemogenesis we created a line of Tle1 null mice. Tle1 null mice are born normally, but become progressively growth retarded by 3 days of life, with only 50% survival by 4 weeks as compared to heterozygous and wild type littermates. Abnormalities are observed in several organs systems including the hematopoietic system. We characterized the hematopoietic system in Tle1 knock out mice between two and 12 weeks of age. The bone marrow cellularity in the Tle1 knock out mice is comparable to the wild type mice at all time points examined. However, frequency of granulocyte macrophage progenitors in bone marrow mononuclear cells is significantly higher in the Tle1 knockout bone marrow compared to heterozygous and wild type mice. The proportion and number of myeloid cells as evidenced by Gr1, Mac1 expression are significantly higher in the bone marrow, spleen and blood of these knockout mice. There were significantly lower B-cells (B220+cells) in the Tle1 knockout mice compared to heterozygous and wild type. In colony forming assays there was a trend towards higher number of CFU-GM (7.66 vs 5), p=0.07) and CFU-M (27.16 vs 12.5, p=0.05) colonies from Tle1 null bone marrow as compared to wild type bone marrow. The spleens from four week and 17 months old Tle1 knockout mice had higher frequency of Gr1-negative, Mac1-positive and F4/80 positive macrophages. We also observed a significantly higher production of the inflammatory cytokines IL6 and TNFafrom peritoneal macrophages harvested from Tle1 null mice as compared to those from wild type mice in response to TLR ligand stimulation. To investigate the potential mechanism of this inhibitory effect of TLE1 on inflammation we demonstrated that TLE1 expression is able to block the nuclear translocation of NFkB in THP1 cells in response to LPS-K12 (p In summary this work demonstrates that the lack of Tle1 expression biases hematopoiesis towards myeloid differentiation, a finding of potential relevance given the inactivation of TLE1 seen in subsets of myeloid malignancies. We further show that inactivation of Tle1 leads to an increase in macrophages primed to release increased inflammatory cytokines. This is notable given the recent observation that TLE1 may modulate the effects of NOD2 in the pathogenesis of Crohn's disease. These Tle1 null mice will allow the investigation of the potential role of TLE1 as a modulator of a variety of other inflammatory diseases. Disclosures: No relevant conflicts of interest to declare.

Abstract 2473 The Groucho/TLE family of corepressors has been described as master regulatory genes during development, affecting multiple cell fate decisions. These proteins bind to a variety of transcription factors and recruit inhibitory proteins to repress transcription. We previously identified TLE1 as a novel tumor suppressor gene that is deleted or methylated in subgroups of acute myeloid leukemia (AML) and other hematological malignancies. We find the loss of Tle1 alone is insufficient to induce leukemia in mice and apparently requires cooperation with additional oncogenes. Our studies, and those from other groups, have shown that over-expression of TLE1 in leukemia cells slows cell cycle progression, colony formation and tumor growth in xenografts, while silencing results in increased cell proliferation. The pathways by which TLE1 affects oncogenesis is unclear, but this gene family is capable of interacting with effectors of Myc, Wnt, Notch, TGFB signaling–prominent pathways dysregulated in malignancies. Myc is important for hematopoietic stem cell proliferation, survival and differentiation and is over-expressed in most AML samples. The TLE homologue Groucho binds and represses Drosophila Myc expression of target genes, thus we postulated that TLE1 could be an important regulator of Myc activity in leukemia. Using hematopoietic progenitor cells from Tle1 knockout and wild-type fetal livers we found that the loss of Tle1 dramatically increased proliferation and serial replating efficiency. Expression of N-Myc by itself in wild type fetal liver cells triggered significant cell death and apoptosis. However, when N-Myc expression was combined with the additional loss of Tle1, not only was N-Myc induced apoptosis inhibited, but a dramatic cell proliferation, well in excess of that seen with Tle1 loss by itself, was seen. Furthermore, mice transplanted with N-Myc transduced hematopoietic cells from Tle1 knockout mice fetal liver developed a more aggressive leukemia, compared to N-Myc transfected wild type mice fetal liver hematopoietic cells, with increased proliferation of leukemic cells as demonstrated by in vitro colony assays and higher secondary transplantability. We extended these studies to a zebrafish model of Rag2-Myc mediated T-ALL. Using these zebrafish we demonstrated over-expression of the TLE homologue, Groucho, completely blocked the initiation and progression of Myc induced leukemia development. Expression of a truncated version of Groucho reduced the initiation of T-ALL and prolonged the survival of fish developing leukemia. These studies demonstrate TLE1 can inhibit the oncogenicity of Myc, and suggests modulation of expression of this gene family may be of importance for a variety of malignancies. Disclosures: No relevant conflicts of interest to declare.

The Schinzel-Giedion syndrome is a rare autosomal recessive condition with typical facies, skeletal manifestations and congenital hydronephrosis. We report an infant with characteristic findings who had bilateral congenital megacalyces. Congenital megacalyces is believed to be a developmental abnormality, occurs in other malformation syndromes and has not previously been described in the Schinzel-Giedion syndrome.

Transducin-like enhancer of split-1 (TLE1) is a novel tumor suppressor gene that is deleted or methylated in subgroups of acute myeloid leukemia (AML) and other hematological malignancies. TLE1 belongs to a family of Gro/TLE proteins that function as corepressors that can bind and potentially inhibit numerous transcription factors known to be involved in development and carcinogenesis including myc, beta-catenin, NF-kB, and TGF-B. In Drosophila Groucho is recognized as a master regulatory gene in development. Our studies and those from other groups have shown that over-expression of TLE1 in leukemia cells slows cell cycle progression, colony formation and tumor growth in xenografts, while silencing results in increased cell proliferation. To further evaluate the role of TLE1 in development and oncogenesis we have knocked out TLE1 in mice. These mice are viable and do not spontaneously develop malignancies in the absence of additional cooperating oncogenes. In Drosophila the TLE homologue Groucho binds and represses Drosophila Myc expression of target genes. N-Myc is important for hematopoietic stem cell proliferation, survival and differentiation and is over-expressed in most AML samples. We postulated that TLE 1 might be an important regulator of N-Myc activity in leukemia and the loss of TLE may accelerate the leukemia development in cooperation with N-Myc. We demonstrate here that the loss of Tle1 cooperates with N-Myc to dramatically increase the serial replating and proliferation of hematopoietic progenitor cells from fetal livers. Mice transplanted with N-Myc transduced hematopoietic cells from the fetal liver of Tle1 knockout mice developed leukemia with a much shorter latency as compared with fetal liver cells from wild type mice. We are currently investigating the mechanisms of this cooperation. This is the first demonstration that Tle1 is a potent modulator of Myc induced tumorigenesis and may have implications in a broader spectrum of malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3113.

The Groucho (Gro)/TLE family of transcriptional co-repressor proteins have been called master regulatory genes based on their interaction with a variety of transcription factors and their critical role in development. The TLEs have also been shown to play major roles in brain and lymphocyte differentiation. We became interested in this gene family after finding two family members, TLE1 and TLE4, localized to the commonly deleted region on chromosome 9q in acute myeloid leukemia (AML). This deletion is tightly associated with t(8;21), and we recently showed loss of TLE1 and TLE4 cooperated with AML1-ETO to affect myeloid cell proliferation and survival, implicating TLE1 and 4 as potential tumor suppressor genes in AML. Based on their known ability to inhibit the function of several signaling pathways and transcription factors including Wnt/b-catenin, NF-kB, AML1, and Pu.1 known to be important in leukemogenesis and hematopoiesis, we undertook a series of experiments to determine whether the TLEs could affect myeloid cell differentiation and proliferation. We showed that expression of either TLE1 or TLE4 was able to induce differentiation in the HL-60 myeloid cell line as demonstrated by morphological changes, increased expression of the myeloid differentiation makers CD11b, CD14 and CD15, downregulated myeloperoxidase activity, as well as increased nitroblue tetrazolium (NBT) staining. Furthermore, when HL-60 cells were induced to differentiate by exposure to all-trans-retinoic acid (ATRA) we observed a 150-fold transient increase in TLE1 message after three days. Knockdown of TLE1 with specific shRNAs partially blocked ATRA-induced differentiation as monitored by CD11b expression, suggesting this burst of TLE1 expression is critical for ATRA induced myeloid cell differentiation. To determine the role of the TLEs in primary human cells, human cord blood cells were sorted, and TLE mRNA levels were determined in progenitor cells (CD34+/CD33−/CD16−/CD15−/CD14−), early myeloid precursors (CD34−/CD33+/CD16−/CD15−CD14−) and mature granulocytes (CD34−/CD33+/CD16+/CD15−CD14−). Lowest levels of TLE1 and TLE4 were found in progenitor cells with a peak of expression in early myeloid precursors. To help determine the significance of these low TLE levels in hematopoietic stem/progenitor cells, we designed shRNAs to simultaneously knockdown both TLE1 and TLE4. Knockdown of TLE1/4 in cord blood cells resulted in a 6.3-fold expansion of CD34+CD38− cells after 4 days’ coculture with M2-10B4 in the presence of hSCF, hFlt3-ligand, and hTPO, as compared with only 2.6-fold expansion of cells transfected with control shRNA. Similarly, using mouse bone marrow cells cultured in vitro in the presence of mIL-3, mIL-6 and mSCF, we found a 2.1-fold increase in mouse bone marrow Lin−/Sca-1+/c-kit+ (LSK) cells transfected with TLE1/4 shRNA as compared to cells transfected with scrambled control shRNA. Our results indicate that modulation of TLE levels is capable of influencing both myeloid differentiation, as well as expansion of the stem cell and/or progenitor population.