Short Arm Of Human Chromosome Research Articles

Silencing XIST on the future active X: Searching human and bovine preimplantation embryos for the repressor.

X inactivation is the means of equalizing the dosage of X chromosomal genes in male and female eutherian mammals, so that only one X is active in each cell. The XIST locus (in cis) on each additional X chromosome initiates the transcriptional silence of that chromosome, making it an inactive X. How the active X in both males and females is protected from inactivation by its own XIST locus is not well understood in any mammal. Previous studies of autosomal duplications suggest that gene(s) on the short arm of human chromosome 19 repress XIST on the active X. Here, we examine the time of transcription of some candidate genes in preimplantation embryos using single-cell RNA sequencing data from human embryos and qRT-PCR from bovine embryos. The candidate genes assayed are those transcribed from 19p13.3-13.2, which are widely expressed and can remodel chromatin. Our results confirm that XIST is expressed at low levels from the future active X in embryos of both sexes; they also show that the XIST locus is repressed in both sexes when pluripotency factors are being upregulated, during the 4-8 cell and morula stages in human and bovine embryos - well before the early blastocyst (E5) when XIST on the inactive X in females starts to be upregulated. Our data suggest a role for DNMT1, UHRF1, SAFB and SAFB2 in XIST repression; they also exclude XACT and other 19p candidate genes and provide the transcriptional timing for some genes not previously assayed in human or bovine preimplantation embryos.

Human chromosome 3p21.3 carries TERT transcriptional regulators in pancreatic cancer

Frequent loss of heterozygosity (LOH) on the short arm of human chromosome 3 (3p) region has been found in pancreatic cancer (PC), which suggests the likely presence of tumor suppressor genes in this region. However, the functional significance of LOH in this region in the development of PC has not been clearly defined. The human telomerase reverse transcriptase gene (hTERT) contributes to unlimited proliferative and tumorigenicity of malignant tumors. We previously demonstrated that hTERT expression was suppressed by the introduction of human chromosome 3 in several cancer cell lines. To examine the functional role of putative TERT suppressor genes on chromosome 3 in PC, we introduced an intact human chromosome 3 into the human PK9 and murine LTPA PC cell lines using microcell-mediated chromosome transfer. PK9 microcell hybrids with an introduced human chromosome 3 showed significant morphological changes and rapid growth arrest. Intriguingly, microcell hybrid clones of LTPA cells with an introduced human chromosome 3 (LTPA#3) showed suppression of mTert transcription, cell proliferation, and invasion compared with LTPA#4 cells containing human chromosome 4 and parental LTPA cells. Additionally, the promoter activity of mTert was downregulated in LTPA#3. Furthermore, we confirmed that TERT regulatory gene(s) are present in the 3p21.3 region by transfer of truncated chromosomes at arbitrary regions. These results provide important information on the functional significance of the LOH at 3p for development and progression of PC.

Generation of induced pluripotent stem cell GZHMCi002-A from peripheral blood mononuclear cells with APOB mutation.

Apolipoprotein (apo) B is a large, amphipathic glycoprotein which plays an important role in human lipoprotein metabolism. The 43-kb APOB gene located on the short arm of human chromosome 2 and consisted of 29 exons, mutations in the APOB gene can give rise to either hypo- or hypercholesterolemia. We used peripheral blood mononuclear cells (PBMCs) from a volunteer carrying the APOB mutation (c.10579C>T, p.Arg3527Trp) located in exon 9 to establish induced pluripotent stem cells (iPSC), which will be an effective means to reveal the key biologically relevant metabolic mechanisms, a powerful tool for medicine selection and related research.

ANRIL and atherosclerosis.

The 3.8-kb-long antisense non-coding RNA at the INK4 locus (ANRIL) is transcribed from the short arm of human chromosome 9 on P21 and is associated with malfunction of the vascular endothelium, vascular smooth muscle cell (VSMC) proliferation/migration/senescence/apoptosis, mononuclear cell adhesion and proliferation, glycolipid metabolism disorder and DNA damage. Hence, ANRIL plays an important role in atherogenesis. Moreover, genome-wide association studies (GWAS) have identified ANRIL as a biomarker that is closely related to coronary heart disease (CHD). The objective of this review was to discuss the pathological mechanism of ANRIL in atherosclerotic development and its significance as a predictor of cardiovascular disease. Review of the PubMed, EMBASE and Cochrane databases for articles demonstrating the roles of ANRIL in the development of atherosclerotic diseases. The abnormal expression of ANRIL is linked to vascular endothelium injury; the proliferation, migration, senescence and apoptosis of VSMCs; mononuclear cell adhesion and proliferation; glycolipid metabolism disorder; DNA damage; and competing endogenous RNAs. Moreover, ANRIL accelerates the progression of CHD by regulating its single nucleotide polymorphisms (SNPs). Considering that ANRIL accelerates atherosclerosis (AS) development and is a risk factor for CHD, it is reasonable for us to explore an efficacious ANRIL-based therapy for AS in CHD.

Biological and Clinical Significance of Human NKRP1A/LLT1 Receptor/Ligand Interactions.

Killer cell lectin-like receptors (KLRs) are C-type lectin-like glycoproteins encoded by genes clustered in the natural killer gene complex (NKC) located on the short arm of human chromosome 12. In addition to the NKG2 subfamily, the NKC includes a less characterized group of genes coding for NKRP1 receptors and their ligands of the C-type lectin (CLEC) subfamily. Among this group, the best recognized is the NKRP1A/LLT1 pair encoded respectively by the KLRB1 and CLEC2D genes. Both molecules are type II transmembrane-signaling glycoproteins with an extracellular C-type lectin domain. NKRP1A is predominantly expressed on NK cells, where it acts as an inhibitory receptor. However, it stimulates T cells, which results in release of IL-17 and inflammatory cytokines. Triggering LLT1 on NK cells stimulates IFN-γ production. Similarly, it stimulates activation of B cells. LLT1 is also expressed by osteoblasts and chondrocytes and inhibits bone degradation. Expression of LLT1 by tumor cells may facilitate their escape from NK cell surveillance. On the other hand, NKRP1A may be involved in activation of T and B lymphocytes in the course of inflammatory reactions and pathogenesis of autoimmune disorders. Thus, the NKRP1A/LLT1 receptor/ligand system appears to be a new therapeutic target that may be useful in the treatment of cancer as well as some autoinflammatory disorders.

Pathogenesis, Diagnostics, and Treatment of Hereditary Haemochromatosis: A 150 Year-Long Understanding of an Iron Overload Disorder

Haemochromatosis is an iron overload disorder that can be inherited or acquired and when diagnosis is delayed, disease progression and death can occur. Iron overload was first described by the French internist Armand Trousseau in 1865 in an article on diabetes in which alterations in skin pigmentations were reported. Some years later, the German pathologist Friedrich Daniel von Recklinghausen coined the term ‘haemochromatosis’ for a metabolic disorder characterised by excess deposition of iron in the tissue. This disorder affects 1 in 200 subjects of Caucasians of Northern European descent. The systemic excess iron build-up condition quickly gained an intense clinical interest. Haemochromatosis can lead to severe pathological symptoms in multiple organs, including the liver, bones, spleen, heart, pancreas, joints, and reproductive organs. With the progress of the disease, hepatic damage predominates. Polymorphisms in several independent genes can lead to haemochromatosis. However, the most widely known haemochromatosis-associated and studied ones are genetic variants in the HFE gene, located on the short arm of human chromosome 6. Early detection and phlebotomy prior to the onset of fibrosis/cirrhosis can reduce morbidity and normalise life expectancy. Consequently, phlebotomy has been accepted for decades as a standard treatment for the reduction of iron load. Nowadays, other methods, such as erythrocytapheresis, therapeutic application of iron chelators and proton pump inhibitors, or hepcidin-targeted therapy, are discussed as alternative personalised treatments of hereditary haemochromatosis. This review focusses on the pathogenesis, diagnosis, and therapy of haemochromatosis.

Long Non-coding RNA ANRIL and Polycomb in Human Cancers and Cardiovascular Disease.

The long non-coding RNA CDKN2B-AS1, commonly referred to as the A ntisense N on-coding R NA in the I NK4 L ocus (ANRIL), is a 3.8-kb-long RNA transcribed from the short arm of human chromosome 9 on p21.3 that overlaps a critical region encompassing three major tumor suppressor loci juxtaposed to the INK4b-ARF-INK4a gene cluster and the methyl-thioadenosine phosphorylase (MTAP) gene. Genome-wide association studies have identified this region with a remarkable and growing number of disease-associated DNA alterations and single nucleotide polymorphisms, which corresponds to increased susceptibility to human disease. Recent attention has been devoted on whether these alterations in the ANRIL sequence affect its expression levels and/or its splicing transcript variation, and in consequence, global cellular homeostasis. Moreover, recent evidence postulates that ANRIL not only can regulate their immediate genomic neighbors in cis, but also has the capacity to regulate additional loci in trans. This action would further increase the complexity for mechanisms imposed through ANRIL and furthering the scope of this lncRNA in disease pathogenesis. In this chapter, we summarize the most recent findings on the investigation of ANRIL and provide a perspective on the biological and clinical significance of ANRIL as a putative biomarker, specifically, its potential role in directing cellular fates leading to cancer and cardiovascular disease.

P01-006 – MEFV mutation detection in Arabic patients

Autoinflammatory diseases are a group of disorders characterized by seemingly unprovoked inflammation in the absence of high-titer autoantibodies or antigen-specific T-cells. Familial Mediterranean fever (FMF) is the archetypal hereditary periodic fever syndrome and autoinflammatory disorder. It is characterized by recurrent self-limiting episodes of fever and painful polyserositis. FMF is an autosomal recessive disorder, with considerable prevalence in specific ethnic groups, namely, non-Ashkenazi Jews, Armenians, Turks and Arabs and the FMF carrier rate can be as high as one in four. The gene responsible for FMF, MEFV, is located on the short arm of human chromosome 16, and was independently identified by two positional cloning consortia. Mutations, as well as, polymorphisms in MEFV are continuously identified. In Arabic FMF patients the spectrum and distribution of MEFV mutations are distinctive and the portion of unidentified mutations is undoubtedly the highest amongst the groups commonly affected by FMF. The comprehensive identification of MEFV mutant alleles among FMF patients is needed for the efficient examination of specific genotype – phenotype correlation patterns and for the development of molecular tools to support the clinical diagnosis.

Tu1130 Simple Endoscopic Score for Crohn's Disease (SES-CD) Predicts Clinical Remission in Patients With Crohn's Disease

Introduction: Centaurin Beta 1 (CENTB1) also named ACAP1, previously identified as a GTPase-activating protein, is a member of the ADP ribosylation factor family [1]. This protein is encoded by a gene located on the short arm of human chromosome 17. A previous study showed that CENTB1 binds to NOD1 and NOD2, and serves as a cytosolic down-regulator of NF-kB activation in response to γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) and muramyl dipeptide (MDP-LD) stimulation in intestinal epithelial cells. No previous have explore the expression of CENTB1 in patients with Ulcerative Colitis (UC). Aim: To study the CENTB1 gene and protein expression in patients with UC. Patients and Methods: We measured the CENTB1 gene expression from colonic biopsies from three groups: 1) Active UC (n=20); 2) Remission UC (n=20); and Healthy control group (n=20). All individuals were assessed for CENTB1 mRNA transcripts levels relative to GAPDH constitutive gene using Real Time Polymerase Chain Reaction using LNA TaqMan® probes from Roche in combination with target gene specific primers: CENTB1 left: atgccacccaacacacact and right: gaaaccccgcagaccttc; and GAPDH left: agccacatcgctcagaca and right: gcccaatacgaccaaatcc for normalization. The detection of CENTB1 protein in tissue was performed by immunohistochemistry. Results: The CENTB1 gene expression was significant up-regulated in colonic mucosa from patients with active UC compared to remission UC and the healthy control group (P= 0.006 and P=0.031 respectively). Also, a marginal increased gene expression of CENTB1 was detected in the UC remission group compared to the healthy control group (P=0.04). In order to confirm the localization of CENTB1 protein in colonic tissue (in situ), we performed a detailed immunohistochemical analysis for CENTB1 in inflamed and noninflamed colonic tissue from UC patients. The percentage of centaurin beta 1 producing cells was higher in UC patients compared with controls. Centaurin beta1+ cells were localized mainly in inflammatory infiltrates, predominantly polymorphonuclear cells, which extended from the adventitia to mucosa, being more abundant in epithelial cells (p=0.04) and adventitia. Conclusion: The CENTB1 was up-regulated in patients with active UC as compared to UC remission and healthy control groups suggesting that it is involved in the inflammatory process.

SY12-3 - Targeting ALK Tyrosine Kinase in Lung Cancer

ABSTRACT In order to discover novel transforming genes in cancer specimens, we developed a sensitive retroviral expression library system for full-length cDNAs. By using this technology coupled with a focus formation assay with mouse 3T3 cells, we could discover, from a specimen of non-small cell lung cancer (NSCLC), a novel oncogenic fusion-type tyrosine kinase EML4-ALK. Both of EML4 and ALK genes are mapped closely on the same short arm of human chromosome 2, but a small inversion involving these two loci, inv(2)(p21p23), leads to fuse the 5'-part of EML4 to the 3′ part of ALK encoding the tyrosine kinase domain. Transgenic mice expressing EML4-ALK in lung epithelial cells developed hundreds of adenocarcinoma nodules in both lungs only at a few weeks after birth, and oral administration of a specific inhibitor to ALK successfully cleared such nodules from the mice, proving in vivo that EML4-ALK is the primary genetic event to trigger cancer, and, therefore, specific inhibitors against EML4-ALK could be a novel and effective drug.Currently, a number of clinical trials with ALK inhibitors are ongoing worldwide, and the result of phase I/II studies with one of such compound, crizotinib, was made public, demonstrating the dramatic therapeutic efficacy of the compound against EML4-ALK-positive NSCLC. Further, crizotinib has just been approved as a therapeutic drug by US FDA in a record breaking speed of drug development. To help individuals with EML4-ALK-positive NSCLC in Japan, we started from early 2009 ‘ALK Lung Cancer Study group (ALCAS)’, a nation-wide diagnostic network for such tumors. Through the ALCAS initiative, we also noticed a patient who was once effectively treated with crizotinib, but later underwent abrupt relapse. Molecular analyses of these specimens led to the discovery of secondary mutations C1156Y and L1196M within EML4-ALK accounting for the observed drug tolerance. Our data thus demonstrate that a subset of lung cancer express previously unidentified fusion kinases that are promising candidates for a therapeutic target as well as for a diagnostic molecular marker for this intractable disorder.

Loss of Hypermethylated in Cancer 1 (HIC1) in Breast Cancer Cells Contributes to Stress-induced Migration and Invasion through β-2 Adrenergic Receptor (ADRB2) Misregulation

The transcriptional repressor HIC1 (Hypermethylated in Cancer 1) is a tumor suppressor gene inactivated in many human cancers including breast carcinomas. In this study, we show that HIC1 is a direct transcriptional repressor of β-2 adrenergic receptor (ADRB2). Through promoter luciferase activity, chromatin immunoprecipitation (ChIP) and sequential ChIP experiments, we demonstrate that ADRB2 is a direct target gene of HIC1, endogenously in WI-38 cells and following HIC1 re-expression in breast cancer cells. Agonist-mediated stimulation of ADRB2 increases the migration and invasion of highly malignant MDA-MB-231 breast cancer cells but these effects are abolished following HIC1 re-expression or specific down-regulation of ADRB2 by siRNA treatment. Our results suggest that early inactivation of HIC1 in breast carcinomas could predispose to stress-induced metastasis through up-regulation of the β-2 adrenergic receptor.

Maternal gametic transmission of translocations or inversions of human chromosome 11p15.5 results in regional DNA hypermethylation and downregulation of CDKN1C expression

Beckwith–Wiedemann syndrome (BWS) is an overgrowth syndrome associated with genetic or epigenetic alterations in one of two imprinted domains on chromosome 11p15.5. Rarely, chromosomal translocations or inversions of chromosome 11p15.5 are associated with BWS but the molecular pathophysiology in such cases is not understood. In our series of 3 translocation and 2 inversion patients with BWS, the chromosome 11p15.5 breakpoints map within the centromeric imprinted domain, 2. We hypothesized that either microdeletions/microduplications adjacent to the breakpoints could disrupt genomic sequences important for imprinted gene regulation. An alternate hypothesis was that epigenetic alterations of as yet unknown regulatory DNA sequences, result in the BWS phenotype. A high resolution Nimblegen custom microarray was designed representing all non-repetitive sequences in the telomeric 33 Mb of the short arm of human chromosome 11. For the BWS-associated chromosome 11p15.5 translocations and inversions, we found no evidence of microdeletions/microduplications. DNA methylation was also tested on this microarray using the HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) assay. This high-resolution DNA methylation microarray analysis revealed a gain of DNA methylation in the translocation/inversion patients affecting the p-ter segment of chromosome 11p15, including both imprinted domains. BWS patients that inherited a maternal translocation or inversion also demonstrated reduced expression of the growth suppressing imprinted gene, CDKN1C in Domain 2. In summary, our data demonstrate that translocations and inversions involving imprinted domain 2 on chromosome 11p15.5, alter regional DNA methylation patterns and imprinted gene expression in cis, suggesting that these epigenetic alterations are generated by an alteration in “chromatin context”.

Genetic predisposition to diffuse panbronchiolitis

Diffuse panbronchiolitis is characterized by chronic inflammation in respiratory bronchioles and sinobronchial infection. The pathophysiology accompanying the persistent bacterial infection is noteworthy for the accumulation of lymphocytes and foamy macrophages around the small airways, for mucus hypersecretion, and for the number of neutrophils in the large airways. Until the establishment of long-term macrolide therapy, the prognosis was generally poor. Case studies of diffuse panbronchiolitis in East Asians, including Japanese, Koreans and Chinese, have frequently been reported, and genetic predisposition to the disease has been assumed in Asians. Immunogenetic studies revealed a strong association with human leukocyte antigen (HLA)-B54 in Japanese, whereas an association with HLA-A11 was reported in Koreans. These findings imply that a major susceptibility gene may be located between the HLA-A and HLA-B loci on the short arm of human chromosome 6. We have recently cloned novel mucin-like genes in this candidate region. In addition to accumulated knowledge of classical HLA genes and mucin genes, further analysis of newly identified genes may provide insights into the pathogenesis of the disease.

Loss of heterozygosity in the short arm of human chromosome 3 in sporadic lung cancer

Introduccion: La perdida de heterocigocidad (LOH) en el brazo corto del cromosoma 3 (3p) humano es un evento frecuente en diferentes tipos de tumores esporadicos, incluyendo cancer de pulmon (CP). Objetivo: Determinar la LOH de 3p en muestras de CP, con 17 marcadores microsatelitales. Metodologia: En un estudio piloto en voluntarios, se recolectaron 13 biopsias de CP (tejido tumoral) y 4 ml de sangre periferica (tejido normal) del mismo paciente, se extrajo el ADN y se realizaron reacciones en cadena de la polimerasa (PCR) con 17 marcadores microsatelitales para analizar LOH. Los fragmentos amplificados se corrieron en geles de poliacrilamida desnaturalizante al 6% y se visualizaron por medio de la coloracion de tincion de plata. El analisis descriptivo se realizo para cada region estudiada en el cromosoma 3p. Resultados: Todos los tumores fueron informativos para uno o mas de los marcadores analizados. Se encontro LOH en uno o mas loci en 11 muestras (84.6%). Los marcadores con mayores LOH fueron UBE1L (23.1%), D3S1317, D3S1300, D3S1284, D3S1274, D3S3049 y D3S1577 con 15.4%. Tres muestras presentaron inestabilidad microsatelital (cambios en la longitud del microsatelite) en diferentes loci. Los porcentajes de LOH para las regiones de 3p fueron: 17.6 % para 3p24-25, 11.6% para 3p21-22, 20% para 3p13-14 y 18.4% para la region 3p12. Conclusiones: Se identificaron regiones cromosomicas con perdida alelica donde es probable que se localicen otros GST involucrados en el desarrollo de CP, diferentes de los ya identificados como VHL, RASSF1A, FHIT y DUTTI, entre otros. Se debe aumentar el numero de muestras y de marcadores para delimitar una region minima e identificar algun gen no descrito implicado en la carcinogenesis de pulmon

Variation and linkage disequilibrium within odorant receptor gene clusters linked to the human major histocompatibility complex

Odorant receptors (OR) are G-protein–coupled receptors that are predominantly expressed in the membrane of olfactory neurons. Members of the two OR gene clusters on the short arm of human chromosome 6 could be involved in major histocompatibility complex (MHC)–associated behavioral traits, such as olfaction-influenced mate selection and cryptic female choice. In this context, OR gene polymorphisms and haplotypes are likely to play an important role. Here we report an investigation of polymorphisms within 12 MHC-linked OR genes in 10 human cell lines. Eight of these OR loci belong to the telomeric, smaller OR gene cluster, whereas four are located centromeric, between the first cluster and the MHC. We also assessed part of this genomic region using sequence data from eight additional cell lines that had previously been sequenced. Thirteen novel OR variants were found through direct DNA sequencing and cloning, in addition to the detection of OR polymorphisms already known, and the number of OR cluster haplotypes could be increased to 21. Two loci belonging to the telomeric cluster ( OR2B8P and OR1F12) were found to exhibit nonfunctional and potentially functional alleles and should therefore be considered as segregating pseudogenes. The results provide a detailed picture regarding polymorphisms and phenotypic variation in an ethnically diverse sample of major histocompatibility complex–linked OR clusters and identify a subregion of unusually pronounced genetic variability. We expand these data by analyzing linkage disequilibrium both within these OR clusters as well as between them and the HLA complex in 11 unrelated HapMap populations. The sequence data described in this paper have been submitted to the GenBank database under the accession numbers GU251059, GU251060, GU251061, GU251062, GU251063, GU251064, GU251065, GU251066, GU251067, GU251068, GU251069, GU251070, GU251071, and GU251072.

Genomic profile of copy number variants on the short arm of human chromosome 8

We evaluated 966 consecutive pediatric patients with various developmental disorders by high-resolution microarray-based comparative genomic hybridization and found 10 individuals with pathogenic copy number variants (CNVs) on the short arm of chromosome 8 (8p), representing approximately 1% of the patients analyzed. Two patients with 8p terminal deletion associated with interstitial inverted duplication (inv dup del(8p)) had different mechanisms leading to the formation of a dicentric intermediate during meiosis. Three probands carried an identical ∼5.0 Mb interstitial duplication of chromosome 8p23.1. Four possible hotspots within 8p were observed at nucleotide coordinates of ∼10.45, 24.32-24.82, 32.19-32.77, and 38.94-39.72 Mb involving the formation of recurrent genomic rearrangements. Other CNVs with deletion- or duplication-specific start or stop coordinates on the 8p provide useful information for exploring the basic mechanisms of complex structural rearrangements in the human genome.

Abstract SY14-04: Discovery and clinical application of EML4-ALK oncogene in lung cancer

Abstract Lung cancer remains the leading cause of cancer deaths worldwide. Conventional chemotherapeutic regimens improve only marginally the outcome of individuals with non-small-cell lung cancer (NSCLC), resulting in a median survival time of <1 year. In order to discover novel transforming genes in lung cancer, we have developed a sensitive retroviral expression library system for full-length cDNAs, and applied this technology to an NSCLC specimen without EGFR mutations. A 3T3 fibroblast-focus formation assay with the library led to the discovery of a novel fusion gene between the echinoderm microtubule-associated protein-like 4 (EML4) and the anaplastic lymphoma kinase (ALK) genes. Oncogenic activity of EML4-ALK requires the N-terminal coiled-coil domain within EML4 that leads to the constitutive dimerization and, thereby, activation of the fusion protein (Soda et al. Nature 448:561). Since both of the EML4 and ALK genes are closely mapped in an opposite direction to the same short arm of human chromosome 2, a small chromosome inversion involving the two genes is likely to be the underlining mechanism for the generation of the gene fusion, which was indeed evidenced by both of FISH and genomic PCR analyses. As in the case for BCR-ABL, PCR-based detection of the EML4-ALK fusion points should be a highly accurate and sensitive diagnostic tool for NSCLC harboring the fusion gene. As expected, RT-PCR readily detected the fusion cDNA even from sputum containing 10 cells/mL of EML4-ALK-positive cells. It should be noted, however, that dozens of variants may be present for EML4-ALK in NSCLC, in which various introns of EML4 is used for the connection to ALK (Mano. Cancer Sci 99:2349, Horn et al. J Clin Oncol 27:4232). Hence, to detect reliably NSCLC with all these variants, multiplex RT-PCR systems should be used in clinical settings, which can capture all in-frame fusions of the two genes (Choi et al. Cancer Res 68:4971, Takeuchi et al. Clin Cancer Res 14:6618, Takeuchi et al. Clin Cancer Res 15:3143). In order to prove the essential role of EML4-ALK in the carcinogenesis of NSCLC, we generated transgenic mice expressing the fusion gene in lung epithelial cells. Interestingly, such mice developed hundreds of adenocarcinoma nodules in both lungs only at a few weeks after birth (Soda et al. PNAS 105:19893). Further, oral administration of a specific inhibitor to ALK successfully cleared such nodules from the mice, proving in vivo that EML4-ALK is the primary genetic event to generate NSCLC with the fusion kinase, and, therefore, specific inhibitors against EML4-ALK could be an effective molecular targeted therapy for the fusion-positive NSCLC. A specific inhibitor to the ALK enzymatic activity (PF-02341066) is already under clinical trials as reported in the 2009 annual meeting of America Society of Clinical Oncology (abstract #3509) (Kwak et al. J Clin Oncol 27:15s suppl; abstr 3509) and of European Cancer Organization and Congress of the European Society for Medical Oncology (abstract #G6). Knowing the marked therapeutic efficacy of the compound, we tried to develop a reliable strategy to diagnose NSCLC with EML4-ALK. Immunohistochemistry (IHC)-based detection of EML4-ALK is highly difficult, probably, owing to the weak activity of the EML4 promoter that drives the expression of EML4-ALK messages. We had thus examined a suitability of commercially available antibodies to ALK for IHC, and successfully developed the intercalated antibody-enhanced polymer (iAEP) method that enables a reliable detection of EML4-ALK among formalin-fixed and paraffin-embedded specimens (Takeuchi et al. Clin Cancer Res 15:3143). The screening results with iAEP and our multiplex RT-PCR were almost 100% concordant between each other, with only a few exceptional cases. From such disconcordant specimens (IHC-positive but RT-PCR-negative), we further discovered a novel fusion of ALK in NSCLC, that is KIF5B-ALK. Using a combination of multiplex RT-PCR and iAEP, we constructed in March 2009 a Japanese nation-wide diagnostic network for NSCLC with ALK-fusions. This “ALK-Lung Cancer Study Group (ALCAS)” initiative has already collected hundreds of NSCLC specimens, and successfully served as a screening system to select individuals to be treated with ALK inhibitors. These data demonstrate that a subset of lung cancer express previously unidentified fusion kinases that are promising candidates for a therapeutic target as well as for a diagnostic molecular marker for this intractable disorder. Citation Format: Hiroyuki Mano. Discovery and clinical application of EML4-ALK oncogene in lung cancer [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY14-04

Simultaneous down-regulation of tumor suppressor genes RBSP3/CTDSPL, NPRL2/G21 and RASSF1A in primary non-small cell lung cancer

BackgroundThe short arm of human chromosome 3 is involved in the development of many cancers including lung cancer. Three bona fide lung cancer tumor suppressor genes namely RBSP3 (AP20 region),NPRL2 and RASSF1A (LUCA region) were identified in the 3p21.3 region. We have shown previously that homozygous deletions in AP20 and LUCA sub-regions often occurred in the same tumor (P < 10-6).MethodsWe estimated the quantity of RBSP3, NPRL2, RASSF1A, GAPDH, RPN1 mRNA and RBSP3 DNA copy number in 59 primary non-small cell lung cancers, including 41 squamous cell and 18 adenocarcinomas by real-time reverse transcription-polymerase chain reaction based on TaqMan technology and relative quantification.ResultsWe evaluated the relationship between mRNA level and clinicopathologic characteristics in non-small cell lung cancer. A significant expression decrease (≥2) was found for all three genes early in tumor development: in 85% of cases for RBSP3; 73% for NPRL2 and 67% for RASSF1A (P < 0.001), more strongly pronounced in squamous cell than in adenocarcinomas. Strong suppression of both, NPRL2 and RBSP3 was seen in 100% of cases already at Stage I of squamous cell carcinomas. Deregulation of RASSF1A correlated with tumor progression of squamous cell (P = 0.196) and adenocarcinomas (P < 0.05). Most likely, genetic and epigenetic mechanisms might be responsible for transcriptional inactivation of RBSP3 in non-small cell lung cancers as promoter methylation of RBSP3 according to NotI microarrays data was detected in 80% of squamous cell and in 38% of adenocarcinomas. With NotI microarrays we tested how often LUCA (NPRL2, RASSF1A) and AP20 (RBSP3) regions were deleted or methylated in the same tumor sample and found that this occured in 39% of all studied samples (P < 0.05).ConclusionOur data support the hypothesis that these TSG are involved in tumorigenesis of NSCLC. Both genetic and epigenetic mechanisms contribute to down-regulation of these three genes representing two tumor suppressor clusters in 3p21.3. Most importantly expression of RBSP3, NPRL2 and RASSF1A was simultaneously decreased in the same sample of primary NSCLC: in 39% of cases all these three genes showed reduced expression (P < 0.05).

Genetics and molecular pathology of Stargardt-like macular degeneration

Stargardt-like macular degeneration (STGD3) is an early onset, autosomal dominant macular degeneration. STGD3 is characterized by a progressive pathology, the loss of central vision, atrophy of the retinal pigment epithelium, and accumulation of lipofuscin, clinical features that are also characteristic of age-related macular degeneration. The onset of clinical symptoms in STGD3, however, is typically observed within the second or third decade of life (i.e., starting in the teenage years). The clinical profile at any given age among STGD3 patients can be variable suggesting that, although STGD3 is a single gene defect, other genetic or environmental factors may play a role in moderating the final disease phenotype. Genetic studies localized the STGD3 disease locus to a small region on the short arm of human chromosome 6, and application of a positional candidate gene approach identified protein truncating mutations in the elongation of very long chain fatty acids-4 gene (ELOVL4) in patients with this disease. The ELOVL4 gene encodes a protein homologous to the ELO group of proteins that participate in fatty acid elongation in yeast. Pathogenic mutations found in the ELOVL4 gene result in altered trafficking of the protein and behave with a dominant negative effect. Mice carrying an Elovl4 mutation developed photoreceptor degeneration and depletion of very long chain fatty acids (VLCFA). ELOVL4 protein participates in the synthesis of fatty acids with chain length longer than 26 carbons. Studies on ELOVL4 indicate that VLCFA may be necessary for normal function of the retina, and the defective protein trafficking and/or altered VLCFA elongation underlies the pathology associated with STGD3. Determining the role of VLCFA in the retina and discerning the implications of abnormal trafficking of mutant ELOVL4 and depleted VLCFA content in the pathology of STGD3 will provide valuable insight in understanding the retinal structure, function, and pathology underlying STGD3 and may lead to a better understanding of the process of macular disease in general.

Synteny mapping of human chromosome 8 loci in cattle

Four genes having homologous loci on the short arm of human chromosome 8 have been mapped to two different bovine syntenic groups. The gene coding for the tissue-type plasminogen activator mapped with GSR, a human chromosome 8 marker, of syntenic group U14 while lipoprotein lipase and the medium and light neurofilament polypeptide genes were shown to be syntenic with the human chromosome 9 marker GGTB2 of syntenic group U18.