Wild-type cDNA Research Articles

PLEKHM2 mutation leads to abnormal localization of lysosomes, impaired autophagy flux and associates with recessive dilated cardiomyopathy and left ventricular noncompaction.

Gene mutations, mostly segregating with a dominant mode of inheritance, are important causes of dilated cardiomyopathy (DCM), a disease characterized by enlarged ventricular dimensions, impaired cardiac function, heart failure and high risk of death. Another myocardial abnormality often linked to gene mutations is left ventricular noncompaction (LVNC) characterized by a typical diffuse spongy appearance of the left ventricle. Here, we describe a large Bedouin family presenting with a severe recessive DCM and LVNC. Homozygosity mapping and exome sequencing identified a single gene variant that segregated as expected and was neither reported in databases nor in Bedouin population controls. The PLEKHM2 cDNA2156_2157delAG variant causes the frameshift p.Lys645AlafsTer12 and/or the skipping of exon 11 that results in deletion of 30 highly conserved amino acids. PLEKHM2 is known to interact with several Rabs and with kinesin-1, affecting endosomal trafficking. Accordingly, patients' primary fibroblasts exhibited abnormal subcellular distribution of endosomes marked by Rab5, Rab7 and Rab9, as well as the Golgi apparatus. In addition, lysosomes appeared to be concentrated in the perinuclear region, and autophagy flux was impaired. Transfection of wild-type PLEKHM2 cDNA into patient's fibroblasts corrected the subcellular distribution of the lysosomes, supporting the causal effect of PLEKHM2 mutation. PLEKHM2 joins LAMP-2 and BAG3 as a disease gene altering autophagy resulting in an isolated cardiac phenotype. The association of PLEKHM2 mutation with DCM and LVNC supports the importance of autophagy for normal cardiac function.

Gene Therapy Restores Hair Cell Stereocilia Morphology in Inner Ears of Deaf Whirler Mice

Hereditary deafness is one of the most common disabilities affecting newborns. Many forms of hereditary deafness are caused by morphological defects of the stereocilia bundles on the apical surfaces of inner ear hair cells, which are responsible for sound detection. We explored the effectiveness of gene therapy in restoring the hair cell stereocilia architecture in the whirlin mouse model of human deafness, which is deaf due to dysmorphic, short stereocilia. Wild-type whirlin cDNA was delivered via adeno-associated virus (AAV8) by injection through the round window of the cochleas in neonatal whirler mice. Subsequently, whirlin expression was detected in infected hair cells (IHCs), and normal stereocilia length and bundle architecture were restored. Whirlin gene therapy also increased inner hair cell survival in the treated ears compared to the contralateral nontreated ears. These results indicate that a form of inherited deafness due to structural defects in cochlear hair cells is amenable to restoration through gene therapy.

Novel P-TEN-induced putative kinase 1 (PINK1) variant in Indian Parkinson’s disease patient

Loss-of-function mutation in PINK1 is known for causing autosomal recessive early onset Parkinsonism accounting approximately 6.5% of PD cases. Recently, PINK1 has also been shown to cause Parkinson’s disease (PD) in eastern India. Present study is aimed to see its contribution in north-Indian PD patients. A total of 106 PD patients and 60 ethnically matched healthy controls were included in the study. All the patients were screened for mutation in PINK1 by direct DNA sequence analysis of the PCR amplicons covering all exons and exon–intron boundaries. Identified novel variant was reconfirmed by DNA sequencing of 10 randomly selected TA clones containing the variant amplicon. In vitro functional assay of the mutant protein was performed by transfecting COS-7 cell line with wild type and mutant (created by site-directed-mutagenesis) cDNA construct of PINK1 fused to N′ terminal GFP followed by western blot analysis. Two potentially pathogenic, one being novel (p.Q267X) and 6 other apparently non-pathogenic variants were identified. Western blot analysis reveals production of truncated PINK1 fusion protein of ∼55kDa in p.Q267X mutant instead of 82/93kDa of wild type PINK1 fusion protein (molecular weight of GFP is ∼27kDa). Our study concludes that PINK1 variants are prevalent for causing Parkinson’s disease (PD) in India, as revealed by the occurrence of 1.8% (2/106) in PD patients from north Indian population. The novel homozygous variant of PINK1 (c.799C>T) reported here is the plausible cause for disease manifestation in this patient. Future study, however, would be helpful to understand the functional mechanism how this premature PINK1 protein (p.Q267X) responds to cellular stress leading to the PD pathophysiology.

A Point Mutation in PDGFRB Causes Autosomal-Dominant Penttinen Syndrome

Penttinen syndrome is a distinctive disorder characterized by a prematurely aged appearance with lipoatrophy, epidermal and dermal atrophy along with hypertrophic lesions that resemble scars, thin hair, proptosis, underdeveloped cheekbones, and marked acro-osteolysis. All individuals have been simplex cases. Exome sequencing of an affected individual identified a de novo c.1994T>C p.Val665Ala variant in PDGFRB, which encodes the platelet-derived growth factor receptor β. Three additional unrelated individuals with this condition were shown to have the identical variant in PDGFRB. Distinct mutations in PDGFRB have been shown to cause infantile myofibromatosis, idiopathic basal ganglia calcification, and an overgrowth disorder with dysmorphic facies and psychosis, none of which overlaps with the clinical findings in Penttinen syndrome. We evaluated the functional consequence of this causative variant on the PDGFRB signaling pathway by transfecting mutant and wild-type cDNA into HeLa cells, and transfection showed ligand-independent constitutive signaling through STAT3 and PLCγ. Penttinen syndrome is a clinically distinct genetic condition caused by a PDGFRB gain-of-function mutation that is associated with a specific and unusual perturbation of receptor function.

TRMT5 Mutations Cause a Defect in Post-transcriptional Modification of Mitochondrial tRNA Associated with Multiple Respiratory-Chain Deficiencies

Deficiencies in respiratory-chain complexes lead to a variety of clinical phenotypes resulting from inadequate energy production by the mitochondrial oxidative phosphorylation system. Defective expression of mtDNA-encoded genes, caused by mutations in either the mitochondrial or nuclear genome, represents a rapidly growing group of human disorders. By whole-exome sequencing, we identified two unrelated individuals carrying compound heterozygous variants in TRMT5 (tRNA methyltransferase 5). TRMT5 encodes a mitochondrial protein with strong homology to members of the class I-like methyltransferase superfamily. Both affected individuals presented with lactic acidosis and evidence of multiple mitochondrial respiratory-chain-complex deficiencies in skeletal muscle, although the clinical presentation of the two affected subjects was remarkably different; one presented in childhood with failure to thrive and hypertrophic cardiomyopathy, and the other was an adult with a life-long history of exercise intolerance. Mutations in TRMT5 were associated with the hypomodification of a guanosine residue at position 37 (G37) of mitochondrial tRNA; this hypomodification was particularly prominent in skeletal muscle. Deficiency of the G37 modification was also detected in human cells subjected to TRMT5 RNAi. The pathogenicity of the detected variants was further confirmed in a heterologous yeast model and by the rescue of the molecular phenotype after re-expression of wild-type TRMT5 cDNA in cells derived from the affected individuals. Our study highlights the importance of post-transcriptional modification of mitochondrial tRNAs for faithful mitochondrial function.

Variable content of von Willebrand factor mutant monomer drives the phenotypic variability in a family with von Willebrand disease

AbstractVon Willebrand disease (VWD) is an inherited bleeding disorder characterized by incomplete penetrance and variable expressivity. We evaluated a 24-member pedigree with VWD type 2 caused by a T>G mutation at position 3911 that predicts a methionine to arginine (M1304R) change in the platelet-binding A1 domain of von Willebrand factor (VWF). This mutation manifests as an autosomal-dominant trait, with clinical and biochemical phenotypic variability among affected individuals, including differences in bleeding tendency and VWF quantity, activity, and multimer pattern. Sequencing of all VWF coding regions in 3 affected individuals did not identify additional mutations. When expressed in heterologous cells, M1304R was secreted in lower quantities, failed to drive formation of storage granules, and was defective in multimerization and platelet binding. When cotransfected in equal quantities with the wild-type complementary DNA, the mutant complementary DNA depressed VWF secretion, although multimerization was only mildly affected. A llama nanobody (AU/VWFa-11) that detects the mutant A1 domain demonstrated highly variable binding to VWF from different affected members, indicating that the VWF contained different percentages of mutant monomers in different individuals. Thus, the observed variability in VWD phenotypes could in part be determined by the extent of mutant monomer incorporation in the final multimer structure of plasma VWF.

Functional and in silico assessment of MAX variants of unknown significance.

The presence of germline mutations affecting the MYC-associated protein X (MAX) gene has recently been identified as one of the now 11 major genetic predisposition factors for the development of hereditary pheochromocytoma and/or paraganglioma. Little is known regarding how missense variants of unknown significance (VUS) in MAX affect its pivotal role in the regulation of the MYC/MAX/MXD axis. In the present study, we propose a consensus computational prediction based on five "state-of-the-art" algorithms. We also describe a PC12-based functional assay to assess the effects that 12 MAX VUS may have on MYC's E-box transcriptional activation. For all but two of these 12 VUS, the functional assay and the consensus computational prediction gave consistent results; we classified seven variants as pathogenic and three as nonpathogenic. The introduction of wild-type MAX cDNA into PC12 cells significantly decreased MYC's ability to bind to canonical E-boxes, while pathogenic MAX proteins were not able to fully repress MYC activity. Further clinical and molecular evaluation of variant carriers corroborated the results obtained with our functional assessment. In the absence of clear heritability, clinical information, and molecular data, consensus computational predictions and functional models are able to correctly classify VUS affecting MAX. A functional assay assesses the effects of MAX VUS over MYC transcriptional activity. A consensus computational prediction and the functional assay show high concordance. Variant carriers' clinical and molecular data support the functional assessment.

Amino Acid Substitutions Improve the Immunogenicity of H7N7HA Protein and Protect Mice against Lethal H7N7 Viral Challenge.

Avian influenza A H7N7/NL/219/03 virus creates a serious pandemic threat to human health because it can transmit directly from domestic poultry to humans and from human to human. Our previous vaccine study reported that mice when immunized intranasally (i.n) with live Bac-HA were protected from lethal H7N7/NL/219/03 challenge, whereas incomplete protection was obtained when administered subcutaneously (s.c) due to the fact that H7N7 is a poor inducer of neutralizing antibodies. Interestingly, our recent vaccine studies reported that mice when vaccinated subcutaneously with Bac-HA (H7N9) was protected against both H7N9 (A/Sh2/2013) and H7N7 virus challenge. HA1 region of both H7N7 and H7N9 viruses are differ at 15 amino acid positions. Among those, we selected three amino acid positions (T143, T198 and I211) in HA1 region of H7N7. These amino acids are located within or near the receptor binding site. Following the selection, we substituted the amino acid at these three positions with amino acids found on H7N9HA wild-type. In this study, we evaluate the impact of amino acid substitutions in the H7N7 HA-protein on the immunogenicity. We generated six mutant constructs from wild-type influenza H7N7HA cDNA by site directed mutagenesis, and individually expressed mutant HA protein on the surface of baculovirus (Bac-HAm) and compared their protective efficacy of the vaccines with Bac-H7N7HA wild-type (Bac-HA) by lethal H7N7 viral challenge in a mouse model. We found that mice immunized subcutaneously with Bac-HAm constructs T143A or T198A-I211V or I211V-T143A serum showed significantly higher hemagglutination inhibition and neutralization titer against H7N7 and H7N9 viruses when compared to Bac-HA vaccinated mice groups. We also observed low level of lung viral titer, negligible weight loss and complete protection against lethal H7N7 viral challenge. Our results indicated that amino acid substitution at position 143 or 211 improve immunogenicity of H7N7HA vaccine against H7N7/NL/219/03 virus.

Abstract S5-05: Defective stalled replication fork repair and predisposition to hereditary breast cancer

Abstract BRCA1 is a tumor suppressor gene, and germ line BRCA1 mutations increase the risk of breast cancer. While all cells with BRCA1 mutations exhibit a heterozygous BRCA1mut/+ genotype, cancer develops primarily in females, often at young ages and affects almost exclusively the breast and ovaries. Why BRCA1 shows such tissue specificity, and how a normal cell in a BRCA1 mutation carrier (BRCA1mut/+) gives rise to invasive tumor cells are largely unknown. To determine whether BRCA1 heterozygosity in cells confers defect in any of the multiple, known, BRCA1 functions is a potentially valuable step in achieving a better understanding of BRCA1 mutation-driven cancer predisposition. Thus, we have analyzed a collection of primary mammary BRCA1mut/+ epithelial cells and skin fibroblasts obtained from BRCA1 mutation carriers for such functions. We, and others have recently shown that BRCA1 exhibits a new DNA damage repair function – i.e. repair of stalled replication forks (SFR). Stalled forks, when not resolved, lead to mutations, or collapse into double strand breaks (DSBs). Both outcomes result in what is commonly referred to as replication stress (RS), which, when chronic, is a driving force behind cancer development. To determine if SFR is defective in normal/healthy breast cells in BRCA1 mutation carriers, and whether this haploinsufficiency results in the kind of genomic changes that lead to cancer, we have now generated 18 primary fibroblast strains from skin punch biopsies and 10 primary mammary epithelial cell (MECs) strains from prophylactic mastectomies performed on BRCA1 mutation carrying women. This collection includes N=23 different BRCA1 mutations, which, together, span almost the entire BRCA1 gene. BRCA1+/+ control MECs were derived from tissue collected during reduction mammoplasties and control fibroblasts were derived from skin punch biopsies from women with no BRCA1 mutation. Our current data shows that BRCA1mut/+ strains exhibited multiple, normal BRCA1 functions, including the support of homologous recombination- type double strand break repair (HR-DSBR), cell cycle- associated checkpoint functions, centrosome number control, spindle pole formation, Slug expression and satellite RNA suppression. By contrast, nearly all strains were defective in the repair of stalled replication forks and in the suppression of fork collapse, i.e. replication stress. These defects were rescued by reconstituting BRCA1 heterozygous cells with wild-type BRCA1 cDNA, indicating that they are a product of BRCA1 haploinsufficiency. In addition, the development of sufficient replication stalling rendered BRCA1mut/+ cells defective in an otherwise intact BRCA1 function, HR-DSBR. No such ‘conditional’ haploinsufficiency was detected in any of the other non-haploinsufficient functions, noted above. Given the importance of replication stress in cancer development and of an HR defect in breast cancer pathogenesis, these defects, when they develop serially, could contribute to the BRCA1 breast cancer development pathway. Finally, given the important role of BRCA2, another hereditary breast cancer gene, in stalled fork stability, a similar analysis for BRCA2mut/+ cells from BRCA2 mutation carriers is currently underway and will also be reported at the meeting. Citation Format: Shailja Pathania, Sangeeta Bade, Morwenna Le Guillou, Karly Burke, Ying Su, David T Ting, Kornelia Polyak, Andrea L Richardson, Jean Feunteun, Judy E Garber, David M Livingston. Defective stalled replication fork repair and predisposition to hereditary breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S5-05.

Epidemiological, clinical and biochemical characterization of the p.(Ala359Asp) SMPD1 variant causing Niemann-Pick disease type B.

Niemann-Pick disease type B (NPDB) is a rare, inherited lysosomal storage disorder that occurs due to variants in the sphingomyelin phosphodiesterase 1 (SMPD1) gene and the resultant deficiency of acid sphingomyelinase (ASM) activity. While numerous variants causing NPDB have been described, only a small number have been studied in any detail. Herein, we describe the frequency of the p.(Ala359Asp) variant in the healthy Chilean population, and determine the haplotype background of homozygous patients to establish if this variant originated from a common founder. Genomic DNA samples from 1691 healthy individuals were analyzed for the p.(Ala359Asp) variant. The frequency of p.(Ala359Asp) was found to be 1/105.7, predicting a disease incidence of 1/44 960 in Chile, higher than the incidence estimated by the number of confirmed NPDB cases. We also describe the clinical characteristics of 13 patients homozygous for p.(Ala359Asp) and all of them had moderate to severe NPDB disease. In addition, a conserved haplotype and shared 280 Kb region around the SMPD1 gene was observed in the patients analyzed, indicating that the variant originated from a common ancestor. The haplotype frequency and mitochondrial DNA analysis suggest an Amerindian origin for the variant. To assess the effect of the p.(Ala359Asp) variant, we transfected cells with the ASM-p.(Ala359Asp) cDNA and the activity was only 4.2% compared with the wild-type cDNA, definitively demonstrating the causative effect of the variant on ASM function. Information on common variants such as p.(Ala359Asp) is essential to guide the successful implementation for future therapies and benefit to patients.

An N-terminal formyl methionine on COX 1 is required for the assembly of cytochrome c oxidase

Protein synthesis in mitochondria is initiated by formylmethionyl-tRNA(Met) (fMet-tRNA(Met)), which requires the activity of the enzyme MTFMT to formylate the methionyl group. We investigated the molecular consequences of mutations in MTFMT in patients with Leigh syndrome or cardiomyopathy. All patients studied were compound heterozygotes. Levels of MTFMT in patient fibroblasts were almost undetectable by immunoblot analysis, and BN-PAGE analysis showed a combined oxidative phosphorylation (OXPHOS) assembly defect involving complexes I, IV and V. The synthesis of only a subset of mitochondrial polypeptides (ND5, ND4, ND1, COXII) was decreased, whereas all others were translated at normal or even increased rates. Expression of the wild-type cDNA rescued the biochemical phenotype when MTFMT was expressed near control levels, but overexpression produced a dominant-negative phenotype, completely abrogating assembly of the OXPHOS complexes, suggesting that MTFMT activity must be tightly regulated. fMet-tRNA(Met) was almost undetectable in control cells and absent in patient cells by high-resolution northern blot analysis, but accumulated in cells overexpressing MTFMT. Newly synthesized COXI was under-represented in complex IV immunoprecipitates from patient fibroblasts, and two-dimensional BN-PAGE analysis of newly synthesized mitochondrial translation products showed an accumulation of free COXI. Quantitative mass spectrophotometry of an N-terminal COXI peptide showed that the ratio of formylated to unmodified N-termini in the assembled complex IV was ∼350:1 in controls and 4:1 in patient cells. These results show that mitochondrial protein synthesis can occur with inefficient formylation of methionyl-tRNA(Met), but that assembly of complex IV is impaired if the COXI N-terminus is not formylated.

Association Between the Single Nucleotide Polymorphism and the Level of Aquaporin-4 Protein Expression in Han and Minority Chinese with Inflammatory Demyelinating Diseases of the Central Nervous System.

The purpose of this study was to determine whether or not aquaporin-4 (AQP4) gene mutations are related to the pathogenesis of inflammatory demyelinating diseases in the central nervous system. Polymorphisms of AQP4 exons 1-5 were determined by sequencing DNA from 67 patients with central nervous system inflammatory demyelinating diseases, including neuromyelitis optica (NMO), multiple sclerosis, recurrent or simultaneous bilateral optic neuritis, and longitudinally extensive transverse myelitis. A plasmid with the identified new missense mutation was constructed, and human embryonic kidney cells (HEK293A) were transfected with either the pEGFP-N1-AQP4-M23 vector (bearing the identified mutated cDNA sequence) or with the plasmid bearing the wild-type AQP4 gene sequence. AQP4 protein expression was analyzed in both experimental groups using Western Blot analysis following protein extraction from transfected cells. A synonymous mutation (rs1839318) was detected on exon 3, and an additional synonymous mutation was detected on the exon 2-2 (rs72557968). Most importantly, a new missense mutation was detected on exon 2-1. According to Western blot analysis, the mutated cDNA sequence yielded increased AQP4 protein expression in comparison with the wild-type cDNA sequence (P < 0.05). AQP4 gene mutations are uncommon, occurring in only 3 out of 67 patients. Although it is possible that the mutations contributed to an increased risk of inflammatory central nervous system disease in these individuals, it is unlikely that mutations are a significant contributor to most patients with NMO spectrum disorders in China.

COQ4 mutations cause a broad spectrum of mitochondrial disorders associated with CoQ10 deficiency

Primary Coenzyme Q10 (CoQ10) deficiencies are rare, clinically heterogeneous disorders caused by recessive mutations in several genes encoding proteins involved in CoQ10 biosynthesis. CoQ10, a lipoidal quinone, is an essential component of the electron transport chain (ETC), shuttling electrons from complex I/II to complex III. By whole exome sequencing we identified five individuals carrying biallelic mutations in COQ4. The precise function of human COQ4 is not known, but it seems to play a structural role in stabilizing a multiheteromeric complex, which contains most of CoQ10 biosynthetic enzymes. The clinical phenotypes of the five subjects varied widely, but four had a prenatal or perinatal onset with early fatal outcome. Two unrelated individuals presented with severe hypotonia, bradycardia, respiratory insufficiency and heart failure; two sisters showed antenatal cerebellar hypoplasia, neonatal respiratory distress syndrome, and epileptic encephalopathy. The fifth subject had early-onset but slowly progressive clinical course, dominated by neurological deterioration with hardly any involvement of other organs. CoQ10 amount was reduced in all available specimens from mutant subjects, often associated with decrease of CoQ10-dependent ETC complex activities and reduced oxygen consumption rate in cultured cells. The pathogenic role of all identified mutations was experimentally validated in a recombinant yeast model: oxidative growth, strongly impaired in strains lacking COQ4, was corrected by expressing a human wild-type COQ4 cDNA but failed to be corrected by expressing COQ4 cDNAs with any of the nucleotide variants identified in affected subjects. COQ4 mutations are responsible for early-onset mitochondrial diseases with heterogeneous clinical presentations associated with CoQ10 deficiency.

CCDC90A (MCUR1) Is a Cytochrome c Oxidase Assembly Factor and Not a Regulator of the Mitochondrial Calcium Uniporter

Mitochondrial calcium is an important modulator of cellular metabolism. CCDC90A was reported to be a regulator of the mitochondrial calcium uniporter (MCU) complex, a selective channel that controls mitochondrial calcium uptake, and hence was renamed MCUR1. Here we show that suppression of CCDC90A in human fibroblasts produces a specific cytochrome c oxidase (COX) assembly defect, resulting in decreased mitochondrial membrane potential and reduced mitochondrial calcium uptake capacity. Fibroblasts from patients with COX assembly defects due to mutations in TACO1 or COX10 alsoshowed reduced mitochondrial membrane potential and impaired calcium uptake capacity, both of which were rescued by expression of the respective wild-type cDNAs. Deletion of fmp32, a homolog of CCDC90A in Saccharomyces cerevisiae, an organism that lacks an MCU, also produces a COX deficiency, demonstrating that the function of CCDC90A is evolutionarily conserved. We conclude that CCDC90A plays a role in COX assembly and does not directly regulate MCU.

The D816V C-Kit Mutation Confers Higher Proliferation Activity By JAK-STAT and Src Family Kinase Pathways Compared to N822K C-Kit Mutation in Core-Binding Factor Acute Myeloid Leukemia

Backgrounds: Acute myeloid leukemia (AML) with the t(8;21)(q22;q22) or inv(16)(p13.1q22)/t(16;16)(p13.1;q22) is defined as core-binding factor AML (CBF-AML) and has been considered an AML group with favorable prognosis. CBF-AML produces chimeric protein inhibiting differentiation of normal hematopoiesis such as AML1-ETO and CBFB-MYHII. These chimeric protein (class II) and mutation of the class I with cell proliferation activity such as c-kit, Ras, and Flt3ITD are strongly associated with leukemogenesis of CBF-AML. In recent years, it has been reported that CBF-AML with c-kit mutations show poor prognosis. The c-kitmutation in CBF-AML has been reported in the extracellular domain of exon 8, the juxtamembrane domain of exons 10 and 11, and the A-loop domain with tyrosine kinase activity of exon 17. Among these mutations, D816V in exon 17 is the most frequent mutation in CBF-AML. Some previous in vitro studies report that the D816V mutation confers higher tumor growth and anti-apoptotic potential compared to mutations in the extracellular domain of exon 8 or in the juxtamembrane domains of exons 10 and 11. Interestingly N822K in exon 17 is reported at frequency similar to D816V mutation in Asia. Our previous report (Leukemia. 2011;25(9):1423) suggested the possibility that the prognosis of CBF-AML with the N822K mutation was different from D816V. This result suggests that D816V and N822K may differ functionally even if this mutation is also in the same A-loop. Therefore, the aim of this study is to clarify a difference of the molecular biologic function for leukemogenesis between D816V and N822K.Method: To analyze the molecular function of D816V and N822K, we generated amphotropic retroviral vector expressing c-kit (wild type, D816V, and N822K). After transduction with human IL3 dependent TF-1 cells, we established c-kit expressed TF-1 cells (TF-1 WT, TF-1 D816V, and TF-1 N822K). In these three cells, we compared the proliferation rate by cell growth assay and analyzed the signal pathways by human microarray (Agilent SurePrint G3 Human Gene Expression 8x60K v2) and Western blotting.Results: Expression and phosphorylation analysis showed that wild type, D816V, and N822K c-kit cDNA were individually transduced to the TF-1 cells. Cell growth assay showed IL3 independent proliferation was observed in TF-1 D816V and TF-1 N822K but not TF-1 WT. The proliferation rate of TF-1 D816V is significantly higher than that of TF-1 N822K (18610±6269 cells vs 4354±1109 cells in day 10, p=0.026). Similar results were obtained in the presence of SCF (p=0.057).Subsequently, we investigated the difference of signal pathway for cell proliferation between the TF-1 D816V and TF-1 N822K by Western blotting. Under the condition were starved from IL3, TF-1 WT showed much higher expression of c-kit protein than those of the TF-1 D816V and TF-1 N822K, but the TF-1 WT hardly induced phosphorylation of it. Whereas the same level of constitutive phosphorylation of c-kit protein were observed in the TF-1 D816V and TF-1 N822K. We investigated about the downstream of phosphorylated c-kit protein such as JAK-STAT, MAP kinase, Src family kinase, and PI3kinase pathways in the TF-1 D816V and TF-1 N822K. We observed a strong induction of phosphorylated STAT3 and Src protein in the TF-1 D816V, but phosphorylated ERK protein of MAP kinase pathway in the TF-1 N822K. Finally we compared gene expression profiling of these cells using microarrays to discover the novel signaling pathways which are specific for each cell. We found Mras and Jak3gene expressions in the TF-1 D816V were five or more times greater than that in the TF-1 N822K. Also similar results were shown by Western blotting.Conclusions: We conclude that the D816V c-kit mutation confers higher proliferation activity by JAK-STAT and Src family kinase compared to N822K c-kit mutation. These findings suggest that biological functions of c-kit mutations differ depending on the mutation site, which may affect responsiveness to treatments. DisclosuresNo relevant conflicts of interest to declare.

Generation of SIN-Lentiviral Vectors for the Treatment of Canavan Disease Using Ex Vivo Transduction of Hematopoietic Target Cells

The autosomal recessive leukodystrophy Canavan Disease is caused by an acquired or hereditary mutation in the Aspartoacylase gene (ASPA) leading to enzymatic dysfunction of a critical component of central nervous system metabolic and osmotic homeostasis. This loss of enzyme function results in demyelination, hydrocephalus, spastic paralysis, seizures, and ultimately premature death. Metabolic treatment for Canavan Disease can slow but not stop disease progression. Gene therapy aimed at replacing the defective ASPA gene has been employed in 13 patients using AAV-2 vectors expressing the human wild-type ASPA cDNA driven by a human Neuron Specific Enolase promoter and delivered via intra-parenchymal injection. Long-term follow-up in these patients revealed minimal clinical impact as although there was measurable physiologic activity of the transgene, it was likely less than what would be sufficient to alter the disease. It has been clearly demonstrated in animal models and in human patients that vector transduced hematopoietic long-term repopulating cells can differentiate into self-renewing microglial populations that can seed the CNS following busulfan treatment. To improve gene delivery to the CNS we have chosen to target the hematopoietic system with SIN-lentiviral vectors expressing wild-type ASPA linked via picornavirus 2A sequence to a GFP reporter gene driven by the highly active MND promoter and flanked by the 650bp cHS4 insulator element. We have produced this vector at laboratory scale with unconcentrated titers in the low 107 t.u./ml range. We have demonstrated transmission of full length vector genome to both cell lines and human CD34+ primary cells. Single cell jurkat transducents were expanded to clonal cell lines which all demonstrated measurable ASPA protein by Western blot and functional ASPA enzyme activity by HPLC assay. Transduction of human mobilized peripheral blood CD34+ cells at an MOI of 26 (MOI 13 x 2 exposures) and plated in methylcellulose for CFU-C assay resulted in 40.6% GFP+ cells by flow cytometry and 0.69 vector genomes/ human genome by real-time PCR. Vector and mock treated cells were transplanted into busulfan conditioned NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice and allowed to engraft. After 12 weeks in vivo, mice were sacrificed and the bone marrow extracted. No difference in overall (hCD45+) or lineage specific (hCD33+, 19+, or 71+) human cell engraftment was observed between the arms indicating a lack of toxicity, with a transduction efficiency of 15.3±7.4% GFP+ hCD45+ cells by flow cytometry and 0.23±0.15 vector genomes/ human genome by real- time PCR. Based on the success of these proof-of-concept experiments, we have produced reporterless versions expressing wild-type and human codon-optimized ASPA. We plan to test these vectors for hematopoietic toxicity and transduction efficiency in human primary cells using the NSG mouse xenotransplant model and to test for functional correction in the Nur7 Canavan Disease mouse model by transducing 5-FU treated Nur7 mouse bone marrow cells and transplanting them into busulfan conditioned Nur7 recipients. If these vectors continue to perform well in in vivo preclinical assays, it is our plan to begin large-scale production with translation into clinical trials. DisclosuresNo relevant conflicts of interest to declare.

Vemurafenib Has Potent Antitumor Activity in Patients with Relapsed/Refractory BRAF Mutant Hairy Cell Leukemia

Background: Although treatment with purine analogs is associated with a high response rate, hairy cell leukemia (HCL) remains incurable with a 30-40% relapse rate. For these patients (pts) and those intolerant to purine analogs, novel therapies are needed. The major finding that BRAFV600E mutations occur in 98% of HCL suggests BRAF as a promising therapeutic target. We therefore designed a phase II trial to (1) determine the clinical efficacy of the BRAF inhibitor vemurafenib in pts with relapsed/ refractory HCL and (2) identify biologic determinants of response and resistance to vemurafenib in HCL. Patients and Methods: Pts with BRAF mutant HCL who were refractory or resistant to purine analogs, or who had ≥2 relapses with an indication for treatment (ANC ≤1.0, HGB ≤10, or PLT ≤100K) were enrolled. Eligible pts received vemurafenib 960mg twice daily for 3 months. Bone marrow (BM) evaluations were performed after 3 months to assess response. Pts with partial (PR) or complete response (CR) with detectable minimal residual disease (MRD) were allowed to receive vemurafenib for up to 3 additional months. The primary endpoint of the study was overall response rate (ORR: CR + PR). Using a Simon’s mini-max two-stage design, if at least 4 of the 19 pts (≥20%) achieve ORR in the first stage, an additional 17 patients will be accrued to the second stage. If 11 or more pts achieve ORR out of the 36 pts, the study would be considered worthy of further investigation. Serial peripheral blood and/or BM samples were collected during the study for quantitative BRAF mutant allele burden, serum cytokine, multiparameter flow cytometry, and targeted next-generation sequencing analysis of a 350-gene panel to detect potential predictors of resistance and identify genes collaborating with BRAF mutations in HCL. Results: 22 pts have been enrolled and 20 pts received treatment. The median age was 61 years (range 44-77), and the median number of prior treatments was 3.5 (range 1-7). 20 pts are evaluable for toxicity and 17 patients for disease response with a median follow up of 10 months (range 2-19). The most common adverse events were rash (40%, Gr1-2), arthralgia (30%, Gr1-2), photosensitivity (20%, Gr1), and pruritis (15%, Gr1). Three pts developed squamous cell carcinoma (SCC), all of whom had previous history of SCC. All patients were able to complete the intended treatment (3 cycles: 11 pts, >3 cycles: 6 pts). All 17 evaluable pts achieved complete hematologic recovery with an overall response rate (ORR) of 100%. 6 pts achieved CR (4 MRD- and 2 MRD+) and 11 pts achieved PR with very minimal disease (Figure 1). Responses were rapid with reduction of circulating hairy cells within 24 hours and normalization of sCD25 within 2-3 weeks (Figure 2). This correlated with dephosphorylation of ERK at 1 month. Several additional inflammatory cytokines correlated with the leukemic cell burden identifying novel tumor markers in HCL including sTNF-R2, sIL-1R2, and sIL4R.Genetic analysis identified a median of 3 (range 0-5) somatic mutations co-existing with the BRAFV600E mutation in HCL including recurrent mutations in MLL2 and CREBBP (Figure 2). Several of these alterations were present as minor clones identifying a clonal architecture in HCL which was not previously appreciated.One pt was found to have de novo resistance to vemurafenib. Genetic analysis identified that this pt’s pretreatment HCL cells harbored a previously undescribed missense mutation in IRS1 (IRS1P1201S) in addition to BRAFV600E mutation. Given prior knowledge that IRS1 (Insulin Receptor Substrate 1) activates both MAP kinase and PI3K-AKT signaling, we compared the effects of expression of wildtype and mutant IRS1 cDNAs on signaling. This revealed robust activation of PI3K-AKT signaling induced by IRS1P1201S-mutant cells relative to wildtype.Conclusions: While longer follow-up is needed to assess the durability of the response, our results indicate that vemurafenib has potent antitumor activity in pts with relapsed/refractory BRAF mutant HCL. These data confirm the MAP kinase pathway as a rational promising therapeutic target in HCL and identify activation of signaling pathways parallel to the MAP kinase pathway as a first mechanism of RAF inhibitor resistance in a hematological malignancy. Subsequent studies will be required to prove that inhibition of B-raf may be the best initial therapy in HCL pts who require treatment. [Display omitted] [Display omitted] DisclosuresPark:Genentech: Research Funding. Off Label Use: Vemurafenib in hairy cell leukemia. Rosen:Novartis: Consultancy.

Complementation of the embryo-lethal T-DNA insertion mutant of AUXIN-BINDING-PROTEIN 1 (ABP1) with abp1 point mutated versions reveals crosstalk of ABP1 and phytochromes

The function of the extracytoplasmic AUXIN-BINDING-PROTEIN1 (ABP1) is largely enigmatic. We complemented a homozygous T-DNA insertion null mutant of ABP1 in Arabidopsis thaliana Wassilewskia with three mutated and one wild-type (wt) ABP1 cDNA, all tagged C-terminally with a strepII-FLAG tag upstream the KDEL signal. Based on in silico modelling, the abp1 mutants were predicted to have altered geometries of the auxin binding pocket and calculated auxin binding energies lower than the wt. Phenotypes linked to auxin transport were compromised in these three complemented abp1 mutants. Red light effects, such as elongation of hypocotyls in constant red (R) and far-red (FR) light, in white light supplemented by FR light simulating shade, and inhibition of gravitropism by R or FR, were all compromised in the complemented lines. Using auxin- or light-induced expression of marker genes, we showed that auxin-induced expression was delayed already after 10 min, and light-induced expression within 60 min, even though TIR1/AFB or phyB are thought to act as receptors relevant for gene expression regulation. The expression of marker genes in seedlings responding to both auxin and shade showed that for both stimuli regulation of marker gene expression was altered after 10-20 min in the wild type and phyB mutant. The rapidity of expression responses provides a framework for the mechanics of functional interaction of ABP1 and phyB to trigger interwoven signalling pathways.

G.O.20: AAV-mediated overexpression of Ubiquilin2 mimics ALS and ALS with dementia in naive mice

Mutations in Ubiquilin2 (UBQLN2) gene have been identified in familial forms of Amyotrophic lateral sclerosis (fALS) and ALS/dementia. Ubiquilin2 pathology was also described in both non-UBQLN2 fALS and in sporadic ALS cases. Ubiquilin2 is involved in degradation of ubiquitinated proteins, and functional studies have shown the formation of abnormal protein aggregation in Ubiquilin2 overexpressing cells. According to the evidence suggesting a toxic gain of function of the mutated Ubiquilin2, we set up a new animal model of UBQLN2-ALS by AAV vector expression of mutant or wild type UBQLN2 in the central nervous system of naive mice. Recombinant AAV10 carrying the human mutant (Pro497His) or wild-type Ubiquilin2 cDNA under control of the phosphoglycerate-kinase (PGK) promoter, were delivered through intracerebroventricular injection in newborn FVB mice. An AAV10 encoding the green fluorescent protein (GFP), injected under the same conditions, was used as control. The expression of human Ubiquilin2 in brain and spinal cord extracts of infected animals was evidenced by western blot, one month after injection. Immunofluorescence analyses demonstrated the presence of Ubiquilin2-positive inclusions in the brain and the spinal cord of mutant Ubiquilin2 overexpressing animals, similarly to ALS/dementia patients. The injected animals displayed a reduced brain size compared to controls, a severe astrogliosis in both the spinal cord and the brain, and a reduced number of ChAT + motor neurons in the whole spinal cord. Ubiquilin2-injected mice had a shortened life span and a body weight loss phenotype; they developed muscle weakness, with loss of muscle mass and strength. Finally, to monitor anxiety, one of the first sign of dementia, mice were subjected to the light/dark box test, which revealed a strong anxious phenotype in Ubiquilin2-injected mice. This model will be useful to further dissect the molecular mechanisms of this complex pathology and to find new therapeutic targets.

Mutations in APOPT1, Encoding a Mitochondrial Protein, Cause Cavitating Leukoencephalopathy with Cytochrome c Oxidase Deficiency

Cytochrome c oxidase (COX) deficiency is a frequent biochemical abnormality in mitochondrial disorders, but a large fraction of cases remains genetically undetermined. Whole-exome sequencing led to the identification of APOPT1 mutations in two Italian sisters and in a third Turkish individual presenting severe COX deficiency. All three subjects presented a distinctive brain MRI pattern characterized by cavitating leukodystrophy, predominantly in the posterior region of the cerebral hemispheres. We then found APOPT1 mutations in three additional unrelated children, selected on the basis of these particular MRI features. All identified mutations predicted the synthesis of severely damaged protein variants. The clinical features of the six subjects varied widely from acute neurometabolic decompensation in late infancy to subtle neurological signs, which appeared in adolescence; all presented a chronic, long-surviving clinical course. We showed that APOPT1 is targeted to and localized within mitochondria by an N-terminal mitochondrial targeting sequence that is eventually cleaved off from the mature protein. We then showed that APOPT1 is virtually absent in fibroblasts cultured in standard conditions, but its levels increase by inhibiting the proteasome or after oxidative challenge. Mutant fibroblasts showed reduced amount of COX holocomplex and higher levels of reactive oxygen species, which both shifted toward control values by expressing a recombinant, wild-type APOPT1 cDNA. The shRNA-mediated knockdown of APOPT1 in myoblasts and fibroblasts caused dramatic decrease in cell viability. APOPT1 mutations are responsible for infantile or childhood-onset mitochondrial disease, hallmarked by the combination of profound COX deficiency with a distinctive neuroimaging presentation.