ABSTRACT
Breast cancer (BC) is the most predominant type of cancer among women. The aim of this study is to find new biomarkers that can help in early detection of BC, especially for those who are too young to be screened using mammography as per guidelines. Using microRNA microarray, we previously showed dysregulation of 74 microRNAs in tumors from early BC patients as compared with normal adjacent tissues, which we were interested in studying in blood circulation. In this study, we investigated the expression of 12 microRNA (miR-21/miR-155/miR-23a/miR-130a/miR-145/miR-425-5p/miR-139-5p/miR-451/miR-195/miR-125b/miR-100, and miR-182) in the plasma of 41 newly diagnosed Lebanese BC patients with early invasive ductal carcinoma as compared with 32 healthy controls. Total RNA was extracted from plasma, and expression levels of miRNA of interest were measured using RT-qPCR followed by statistical analysis; miR-21, miR-155, miR-23a, miR-130a, miR-145, miR-425-5p, and miR-139-5p were significantly upregulated and miR-451 was significantly downregulated, in the plasma of BC patients as compared with healthy controls. The positively correlated miR-23a, miR-21, and miR-130a had a high diagnostic accuracy (86%). Importantly, the combination of miR-145/miR-425-5p/miR-139-5p/miR-130a scored the highest diagnostic accuracy of 95% with AUC = 0.97 (sensitivity 97% and specificity 91%). MicroRNAs are promising non-invasive diagnostic biomarkers for early-stage BC with the panel of miR-145/miR-425-5p/miR-139-5p/miR-130a having the highest diagnostic accuracy.
Subject(s)
Biomarkers, Tumor/blood , Biomarkers, Tumor/genetics , Breast Neoplasms/blood , Breast Neoplasms/diagnosis , Circulating MicroRNA/blood , Circulating MicroRNA/genetics , Gene Expression Profiling , Adult , Aged , Aged, 80 and over , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Female , Gene Expression Regulation, Neoplastic , Humans , Middle Aged , Neoplasm Staging , Statistics, Nonparametric , Young AdultABSTRACT
Diacylglycerol (DAG) kinases (DGKs) are a family of enzymes that convert DAG to phosphatidic acid (PA), the physiologic functions of which have been poorly defined. We report here that DGK alpha and zeta synergistically promote T cell maturation in the thymus. Absence of both DGKalpha and zeta (DGKalpha(-/-)zeta(-/-)) results in a severe decrease in the number of CD4(+)CD8(-) and CD4(-)CD8(+) single-positive thymocytes correlating with increased DAG-mediated signaling. Positive selection, but not negative selection, is impaired in DGKalpha(-/-)zeta(-/-) mice. The developmental blockage in DGKalpha(-/-)zeta(-/-) mice can be partially overcome by treatment with PA. Furthermore, decreased DGK activity also promotes thymic lymphomagenesis accompanying elevated Ras and Erk1/2 activation. Our data demonstrate a synergistic and critical role of DGK isoforms in T cell development and tumor suppression, and indicate that DGKs not only terminate DAG signaling but also initiate PA signaling in thymocytes to promote positive selection.
Subject(s)
Cell Differentiation/immunology , Diacylglycerol Kinase/metabolism , Lymphoma/immunology , Lymphoma/pathology , T-Lymphocytes/cytology , T-Lymphocytes/immunology , Animals , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/immunology , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Diacylglycerol Kinase/deficiency , Diacylglycerol Kinase/genetics , Enzyme Activation , Female , Isoenzymes/metabolism , Lymphoma/enzymology , Lymphoma/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Thymus Gland/enzymology , Tissue Culture Techniques , ras Proteins/metabolismABSTRACT
PURPOSE: The aim of our study was to determine the prevalence of amblyopia risk factors in children visiting the American University of Beirut Medical Center (AUBMC) using automated vision screening. METHODS: This was a hospital-based screening of 1102 children aged between 2 and 6 years. Vision screening was performed using PlusoptiX S12 over 2 years (2018-2020). The need for referral to a pediatric ophthalmologist was based on the amblyopia risk factors set forth by the American Association for Pediatric Ophthalmology and Strabismus. Referred patients underwent a comprehensive eye examination. RESULTS: A total of 1102 children were screened, 63 were referred for amblyopia risk factors (5.7%); 37/63 (59%) underwent comprehensive eye examination and 73% were prescribed glasses. Of the non-referred group of children, 6.35% had astigmatism, 6.25% were hyperopic and 3.27% were myopic. The refractive errors observed among the examined patients were distributed as follows: 41% astigmatism, 51% hyperopia, and 8% myopia; amblyopia was not detected. Refractive amblyopia risk factors were associated with the presence of systemic disorders. Bland-Altman plots showed most of the differences to be within limits of agreement. CONCLUSION: Using an automated vision screener in a hospital-based cohort of children aged 2 to 6 years, the rate of refractive amblyopia risk factors was 5.7%. Hyperopia was the most commonly encountered refractive error and children with systemic disorders were at higher risk.
Subject(s)
Amblyopia , Vision Screening , Child , Child, Preschool , Humans , Male , Prevalence , Referral and Consultation , Risk FactorsABSTRACT
PURPOSE: To detect eye tracking abnormalities in children with strabismus in the absence or presence of amblyopia. METHODS: A total of 100 patients aged 7 to 17 years were enrolled prospectively for 2 years from the pediatric ophthalmology clinic of the American University of Beirut Medical Center: 50 children with strabismus (including 24 with amblyopia) and 50 age- and gender-matched controls. Eye tracking with different paradigms was performed. RESULTS: Mean age was 10.66 ± 2.90 years in the strabismus group and 10.02 ± 2.75 years in the control group. Demographic characteristics were similar with respect to vision, gender, and refraction. Four paradigms were tested using the eye tracker: (1) distance/near paradigm: patients with strabismus showed a lower fixation count and longer fixation at both distances and a tendency for decreased latency and percentage of fixation in distant elements; (2) reading paradigm: the strabismus group had a higher fixation count and duration, especially those without amblyopia; (3) location identification paradigm: strabismus group without amblyopia fixated less and with shorter duration on the most flagrant element; and (4) video paradigm: no differences in eye movements were noted. CONCLUSIONS: Significant eye movement deficits were demonstrated in patients with strabismus compared to controls while reading text and identifying prominent elements in a crowded photograph. This was significant in the non-amblyopic subgroup. [J Pediatr Ophthalmol Strabismus. 2019;56(5):297-304.].
Subject(s)
Amblyopia/physiopathology , Eye Movements/physiology , Strabismus/physiopathology , Visual Acuity , Adolescent , Age Distribution , Amblyopia/complications , Amblyopia/epidemiology , Child , Female , Follow-Up Studies , Humans , Incidence , Male , Prospective Studies , Strabismus/complications , Strabismus/epidemiology , United States/epidemiology , Vision, Binocular/physiologyABSTRACT
Autism spectrum disorder (ASD) is characterized by ritualistic-repetitive behaviors and impaired verbal/non-verbal communication. Many ASD susceptibility genes implicated in neuronal pathways/brain development have been identified. The Lebanese population is ideal for uncovering recessive genes because of shared ancestry and a high rate of consanguineous marriages. Aims here are to analyze for published ASD genes and uncover novel inherited ASD susceptibility genes specific to the Lebanese. We recruited 36 ASD families (ASD: 37, unaffected parents: 36, unaffected siblings: 33) and 100 unaffected Lebanese controls. Cytogenetics 2.7 M Microarrays/CytoScan™ HD arrays allowed mapping of homozygous regions of the genome. The CNTNAP2 gene was screened by Sanger sequencing. Homozygosity mapping uncovered DPP4, TRHR, and MLF1 as novel candidate susceptibility genes for ASD in the Lebanese. Sequencing of hot spot exons in CNTNAP2 led to discovery of a 5 bp insertion in 23/37 ASD patients. This mutation was present in unaffected family members and unaffected Lebanese controls. Although a slight increase in number was observed in ASD patients and family members compared to controls, there were no significant differences in allele frequencies between affecteds and controls (C/TTCTG: γ2 value = 0.014; p = 0.904). The CNTNAP2 polymorphism identified in this population, hence, is not linked to the ASD phenotype.
Subject(s)
Autism Spectrum Disorder/genetics , Dipeptidyl Peptidase 4/genetics , Membrane Proteins/genetics , Nerve Tissue Proteins/genetics , Proteins/genetics , Receptors, Thyrotropin-Releasing Hormone/genetics , Adolescent , Cell Cycle Proteins , Child , Child, Preschool , Consanguinity , DNA-Binding Proteins , Female , Genetic Predisposition to Disease , Heredity , Homozygote , Humans , Lebanon , Male , Mutagenesis, Insertional , Oligonucleotide Array Sequence Analysis , Pedigree , Sequence Analysis, DNA , Young AdultABSTRACT
The neuronal ceroid lipofuscinoses (NCLs) are a heterogeneous group of autosomal recessive neurodegenerative diseases comprising Batten and other related diseases plus numerous variants. They are characterized by progressive neuronal cell death. The CLN6 gene was recently identified, mutations in which cause one of the variant late infantile forms of NCL (vLINCL). We describe four novel mutations in the CLN6 gene. This brings the total number of CLN6 mutations known to 11 in 38 families. This suggests that the CLN6 gene may be highly mutable. An American patient of Irish/French/Native American origin was heterozygous for a 4-bp insertion (c.267_268insAACG) in exon 3. The other allele had a point mutation (c.898T>C) in exon 7 resulting in a W300R amino acid change. Two Trinidadian siblings of Indian origin were homozygous for a mutation at the 5' donor splice site of exon 4 (IVS4+1G>T), affecting the first base of the invariant GT at the beginning of intron 4. The fourth novel mutation, a double deletion of 4 bp and 1 bp in exon 7 (c.829_832delGTCG;c.837delG), was identified in a Portuguese patient heterozygous for the I154del Portuguese CLN6 mutation. Four of the 11 mutations identified are in exon 4. Three Portuguese patients with clinical profiles similar to CLN6 patients without defects in CLN6 or other known NCL genes are described. We conclude the following: 1) the CLN6 gene may be a highly mutable gene; 2) exon 4 must code for a segment of the protein crucial for function; 3) vLINCL disease in Portugal is genetically heterogeneous; 4) the I154del accounts for 81.25% of affected CLN6 Portuguese alleles; and 5) three vLINCL Portuguese patients may have defects in a new NCL gene.
Subject(s)
Membrane Proteins/genetics , Neuronal Ceroid-Lipofuscinoses/genetics , Base Sequence , DNA/chemistry , DNA/genetics , DNA Mutational Analysis , Humans , Infant , MutationABSTRACT
Afebrile seizures associated with rotavirus gastroenteritis, respiratory syncytial virus bronchiolitis, influenza infection, asthma, blood transfusions, and intake of a number of drugs (including theophylline, cephalosporins, metronidazole, and acyclovir) with therapeutic drug levels are uncommonly encountered in clinical practice. Reviewed here are the incidence, etiology, clinical presentation, types, diagnosis, associated electroencephalographic changes, and cranial magnetic resonance imaging findings in the literature, as well as management and prognosis of these seizures.
Subject(s)
Seizures/etiology , Anti-Bacterial Agents/adverse effects , Anti-Infective Agents/adverse effects , Antiviral Agents/adverse effects , Asthma/complications , Asthma/epidemiology , Brain/drug effects , Brain/physiopathology , Bronchiolitis/complications , Child , Gastroenteritis/complications , Humans , Influenza, Human/complications , Influenza, Human/epidemiology , Metronidazole/adverse effects , Respiratory Syncytial Virus Infections/complications , Rotavirus Infections/complications , Seizures/chemically induced , Seizures/physiopathology , Theophylline/adverse effects , Transfusion Reaction , Vasodilator Agents/adverse effectsABSTRACT
Juvenile neuronal ceroid lipofuscinosis (JNCL) belongs to the neuronal ceroid lipofuscinoses characterized by blindness/seizures/motor/cognitive decline and early death. JNCL is caused by CLN3 gene mutations that negatively modulate cell growth/apoptosis. CLN3 protein (CLN3p) localizes to Golgi/Rab4-/Rab11-positive endosomes and lipid rafts, and harbors a galactosylceramide (GalCer) lipid raft-binding domain. Goals are proving CLN3p participates in GalCer transport from Golgi to rafts, and GalCer deficits negatively affect cell growth/apoptosis. GalCer/mutant CLN3p are retained in Golgi, with CLN3p rescuing GalCer deficits in rafts. Diminishing GalCer in normal cells by GalCer synthase siRNA negatively affects cell growth/apoptosis. GalCer restores JNCL cell growth. WT CLN3p binds GalCer, but not mutant CLN3p. Sphingolipid content of rafts/Golgi is perturbed with diminished GalCer in rafts and accumulation in Golgi. CLN3-deficient raft vesicular structures are small by transmission electron microscopy, reflecting altered sphingolipid composition of rafts. CLN1/CLN2/CLN6 proteins bind to lysophosphatidic acid/sulfatide, CLN6/CLN8 proteins to GalCer, and CLN8 protein to ceramide. Sphingolipid composition/morphology of CLN1-/CLN2-/CLN6-/CLN8- and CLN9-deficient rafts are altered suggesting changes in raft structure/lipid stoichiometry could be common themes underlying these diseases.
Subject(s)
Galactosylceramides/metabolism , Membrane Glycoproteins/metabolism , Membrane Lipids/metabolism , Membrane Microdomains/metabolism , Molecular Chaperones/metabolism , Neuronal Ceroid-Lipofuscinoses/metabolism , Animals , Apoptosis , Cell Line , Cell Proliferation , Cells, Cultured , Endoplasmic Reticulum/metabolism , Fibroblasts/metabolism , Galactosylceramides/deficiency , Golgi Apparatus/metabolism , Humans , Membrane Glycoproteins/genetics , Membrane Microdomains/ultrastructure , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Knockout , Molecular Chaperones/genetics , Mutation , Neuronal Ceroid-Lipofuscinoses/genetics , Neuronal Ceroid-Lipofuscinoses/pathology , Protein Binding , Protein Transport , Subcellular Fractions , Sulfoglycosphingolipids/metabolism , Tripeptidyl-Peptidase 1ABSTRACT
The neuronal ceroid lipofuscinoses are pediatric neurodegenerative diseases with common clinical features. Of the nine clinical variants (CLN1-CLN9), six have been genetically identified. Most variants manifest cell death and dysregulated sphingolipid metabolism, suggesting the proteins defective in these disorders may interact along one pathway. NCL patient-derived cell lines exhibit cell growth and apoptotic defects that reverse following transfection with the wild-type gene. The membrane-bound proteins CLN3, CLN6, and CLN8 complement each other, as do CLN1 and CLN2 proteins, with respect to growth and apoptosis. The CLN2 protein also corrects growth and apoptosis in CLN3-, CLN6-, and CLN8-deficient cell lines. Neither CLN1-deficient nor CLN2-deficient growth defects are corrected by CLN3, CLN6, and CLN8 proteins. CLN2, CLN3, CLN6, and CLN8 proteins co-immunoprecipitate and co-localize to early and/or recycling endosomes and lipid rafts. Additionally, CLN2p and CLN1p co-immunoprecipitate. The work presented supports interactions between NCL proteins occurring at multiple points along one pathway.
Subject(s)
Neuronal Ceroid-Lipofuscinoses/metabolism , Signal Transduction , Animals , Apoptosis , COS Cells , Cell Line , Chlorocebus aethiops , Humans , Immunoprecipitation , Membrane Glycoproteins/analysis , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Biological , Neuronal Ceroid-Lipofuscinoses/enzymology , Neuronal Ceroid-Lipofuscinoses/genetics , Time Factors , Transfection , Tripeptidyl-Peptidase 1ABSTRACT
A new variant of a group of pediatric neurodegenerative diseases known as neuronal ceroid lipofuscinosis (NCL) or Batten disease has been identified. It is termed CLN9-deficient. CLN9-deficient fibroblasts have a distinctive phenotype of rapid growth and increased apoptosis and diminished levels of ceramide, dihydroceramide, and sphingomyelin. Transfection with CLN8 but not other NCL genes corrected growth and apoptosis in CLN9-deficient cells, although the entire CLN8 sequence was normal. CLN8 is one of the TRAM-Lag1-CLN8 proteins containing a Lag1 motif. The latter imparts (dihydro)ceramide synthase activity to yeast cells. Transfection with the yeast gene Lag1 Sc and the human homolog LASS1 increased ceramide levels and partially corrected growth and apoptosis in CLN9-deficient cells. LASS2,-4,,-5, and -6 also corrected growth and apoptosis. Dihydroceramide levels and dihydroceramide synthase activity were markedly diminished in CLN9-deficient cells. Sequencing of LASS1, LASS2, LASS4, LASS5, and LASS6 genes was normal, and expression levels were increased or normal in CLN9-deficient cells by reverse transcription-PCR. N-(4-Hydroxyphenyl)retinamide (4-HPR), a dihydroceramide synthase activator, corrected growth and apoptosis and increased dihydroceramide synthase activity. Ceramide levels dropped further, and there was no increase in de novo ceramide synthesis, probably due to the effects of 4-HPR as activator of dihydroceramide synthase and inhibitor of dihydroceramide desaturase. Fumonisin B1, a dihydroceramide synthase inhibitor, exaggerated the CLN9-deficient phenotype of accelerated growth, decreased ceramide and increased apoptosis. This was neutralized by 4-HPR. We conclude that the CLN9 protein may be a regulator of dihydroceramide synthase and that 4-HPR could be developed as a treatment for CLN9-deficient patients.
Subject(s)
Membrane Proteins/physiology , Neuronal Ceroid-Lipofuscinoses/metabolism , Oxidoreductases/metabolism , Apoptosis , Cell Line , Cell Proliferation , Ceramides/analysis , Fenretinide/pharmacology , Fibroblasts/pathology , Fumonisins/pharmacology , Humans , Membrane Proteins/genetics , Neuronal Ceroid-Lipofuscinoses/enzymology , Neuronal Ceroid-Lipofuscinoses/pathology , Saccharomyces cerevisiae Proteins/genetics , Sphingosine N-AcyltransferaseABSTRACT
Juvenile Batten disease (JNCL) is an autosomal recessive disease that results from mutations in the CLN3 gene. The wild-type CLN3 gene coding sequence has 15 exons, and the translated protein consists of 438 amino acids. The most commonly observed mutation is a 1.02 kb deletion in the genomic DNA. This deletion results in a truncated protein due to the loss of amino acids 154-438, and the introduction of 28 novel amino acids at the c-terminus. We demonstrate that, compared to normal controls, CLN3-deficient immortalization of lymphoblasts homozygous for this deletion grow at a slower rate, and show increased sensitivity to etoposide-induced apoptosis, supporting the notion that CLN3 may negatively regulate apoptosis. Using immortalized JNCL lymphoblast cell lines as a model system, we assess the effects of specific CLN3 mutations on cell growth rates and protection from etoposide-induced apoptosis. Protection from etoposide-induced apoptosis occurs and the cell growth rate is restored following transfection of JNCL lymphoblasts with mutant CLN3 cDNA that includes exons 11 or 13. We show that deletion of the glycosylation sites 71NQSH74 and 310NTSL313, and also mutations within the highly conserved amino acid stretches 184WSSGTGGAGLLG195, 291VYFAE295 and 330VFASRSSL337, result in slowed growth and susceptibility to apoptosis.
Subject(s)
Amino Acid Motifs/genetics , Apoptosis/genetics , Cell Division/genetics , Membrane Glycoproteins , Molecular Chaperones , Proteins/genetics , Apoptosis/drug effects , Cells, Cultured , DNA/biosynthesis , Etoposide/pharmacology , Glycosylation , Humans , Mutation , Neuronal Ceroid-Lipofuscinoses/genetics , Nucleic Acid Synthesis Inhibitors/pharmacology , Protein BiosynthesisABSTRACT
Juvenile neuronal ceroid lipofuscinosis (JNCL) is due to mutations in the CLN3 gene. We previously determined that CLN3 protein harbors a highly conserved motif, VYFAE, necessary for its impact on cell growth and apoptosis. Using molecular modeling we demonstrated that this motif is embedded in a stretch of amino acids that is homologous to and structurally compatible with a galactosylceramide (GalCer) binding domain. This domain is present in the V3 loop of the HIV-1 gp120 envelope protein, beta-amyloid protein, and the infectious form of prionic protein, and defines a binding site for lipid rafts. We determined the subcellular localization of CLN3 in different cell systems including human neurons, primary rat hippocampal neurons, normal human fibroblasts, and JNCL fibroblasts homozygous for the 1.02 kb deletion in genomic DNA. Wild-type CLN3 protein was present within Golgi, lipid rafts in the plasma membrane, and early recycling endosomes, but not late endosomes/lysosomes. Wild-type CLN3 internalized from the plasma membrane to the Golgi via Rab4- and Rab11-positive recycling endosomes. Wild-type CLN3 co-localized with GalCer in the Golgi and in lipid rafts at the plasma membrane in normal cells. Neither mutant CLN3 protein nor GalCer were found at the plasma membrane in JNCL fibroblasts. Mutant CLN3p was retained within the Golgi and partially mis-localized to lysosomes, failing to reach recycling endosomes, plasma membrane, or lipid rafts. These studies identify a novel CLN3 domain that may dictate localization and function of CLN3.
Subject(s)
Endosomes/metabolism , Galactosylceramides/metabolism , Golgi Apparatus/metabolism , Membrane Glycoproteins/metabolism , Membrane Microdomains/metabolism , Molecular Chaperones/metabolism , Animals , Cells, Cultured , Endosomes/chemistry , Fibroblasts/cytology , Fibroblasts/metabolism , Fluorescent Dyes/metabolism , Golgi Apparatus/chemistry , Humans , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/genetics , Membrane Microdomains/chemistry , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Neuronal Ceroid-Lipofuscinoses/metabolism , Neurons/cytology , Neurons/metabolism , Protein Structure, Tertiary , Protein Transport/physiology , Rats , rab GTP-Binding Proteins/metabolism , rab4 GTP-Binding Proteins/metabolismABSTRACT
Multiple gene defects cause Batten disease. Accelerated apoptosis accounts for neurodegeneration in the late infantile and juvenile forms that are due to defects in the CLN3 and CLN2 genes. Extensive neuronal death is seen in CLN2- and CLN3-deficient human brain as well as in CLN6-deficient sheep brain and retina. When neurons in late infantile and juvenile brain survive, they manage to do so by upregulating the neuroprotective molecule Bcl-2. The CLN3 gene has antiapoptotic properties at the molecular level. We show that the CLN2 gene is neuroprotective: it enhances growth of NT2 cells and maintains survival of human postmitotic hNT neurons. Conversely, blocking CLN3 or CLN2 expression in hNT neurons with adenoviral antisense-CLN3 or antisense-CLN2-AAV2 constructs causes apoptosis. The drug flupirtine is a triaminopyridine derivative that acts as a nonopioid analgesic. Flupirtine upregulates Bcl-2, increases glutathione levels, activates an inwardly rectifying potassium channel, and delays loss of intermitochondrial membrane calcium retention capacity. We show that flupirtine aborts etoposide-induced apoptosis in CLN1-, CLN2-, CLN3-, and CLN6-deficient as well as normal lymphoblasts. Flupirtine also prevents the death of CLN3- and CLN2-deficient postmitotic hNT neurons at the mitochondrial level. We show that a mechanism of neuroprotection exerted by flupirtine involves complete functional antagonism of N-methyl-D-aspartate or N-methyl-D-aspartate-induced neuronal apoptosis. Flupirtine may be useful as a drug capable of halting the progression of neurodegenerative diseases caused by dysregulated apoptosis.
Subject(s)
Aminopyridines/pharmacology , Apoptosis/drug effects , Membrane Glycoproteins , Molecular Chaperones , Neuronal Ceroid-Lipofuscinoses/drug therapy , Neurons/cytology , Neuroprotective Agents/pharmacology , Peptide Hydrolases/genetics , Aminopeptidases , Cell Division/drug effects , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases , Endopeptidases , Gene Expression , Humans , In Situ Nick-End Labeling , In Vitro Techniques , Lymphocytes/cytology , Lymphocytes/drug effects , Membrane Proteins/genetics , Mitosis , Neuronal Ceroid-Lipofuscinoses/genetics , Neuronal Ceroid-Lipofuscinoses/pathology , Neurons/drug effects , Neurons/physiology , Proteins/genetics , RNA, Messenger/analysis , Serine Proteases , Teratocarcinoma , Thiolester Hydrolases , Transfection , Tripeptidyl-Peptidase 1 , Tumor Cells, CulturedABSTRACT
The CLN6 gene that causes variant late-infantile neuronal ceroid lipofuscinosis (vLINCL), a recessively inherited neurodegenerative disease that features blindness, seizures, and cognitive decline, maps to 15q21-23. We have used multiallele markers spanning this approximately 4-Mb candidate interval to reveal a core haplotype, shared in Costa Rican families with vLINCL but not in a Venezuelan kindred, that highlighted a region likely to contain the CLN6 defect. Systematic comparison of genes from the minimal region uncovered a novel candidate, FLJ20561, that exhibited DNA sequence changes specific to the different disease chromosomes: a G-->T transversion in exon 3, introducing a stop codon on the Costa Rican haplotype, and a codon deletion in exon 5, eliminating a conserved tyrosine residue on the Venezuelan chromosome. Furthermore, sequencing of the murine homologue in the nclf mouse, which manifests recessive NCL-like disease, disclosed a third lesion-an extra base pair in exon 4, producing a frameshift truncation on the nclf chromosome. Thus, the novel approximately 36-kD CLN6-gene product augments an intriguing set of unrelated membrane-spanning proteins, whose deficiency causes NCL in mouse and man.