ABSTRACT
Canine leukocyte adhesion deficiency (CLAD) provides a unique large animal model for testing new therapeutic approaches for the treatment of children with leukocyte adhesion deficiency (LAD). In our CLAD model, we examined two different fragments of the human elongation factor 1alpha (EF1alpha) promoter (EF1alphaL, 1189 bp and EF1alphaS, 233 bp) driving the expression of canine CD18 in a self-inactivating (SIN) lentiviral vector. The EF1alphaS vector resulted in the highest levels of canine CD18 expression in CLAD CD34(+) cells in vitro. Subsequently, autologous CD34(+) bone marrow cells from four CLAD pups were transduced with the EF1alphaS vector and infused following a non-myeloablative dose of 200 cGy total-body irradiation. None of the CLAD pups achieved levels of circulating CD18(+) neutrophils sufficient to reverse the CLAD phenotype, and all four animals were euthanized because of infections within 9 weeks of treatment. These results indicate that the EF1alphaS promoter-driven CD18 expression in the context of a RRLSIN lentiviral vector does not lead to sufficient numbers of CD18(+) neutrophils in vivo to reverse the CLAD phenotype when used in a non-myeloablative transplant regimen in dogs.
Subject(s)
CD18 Antigens/genetics , Genetic Therapy/methods , Genetic Vectors , Lentivirus , Leukocyte-Adhesion Deficiency Syndrome/therapy , Peptide Elongation Factor 1/genetics , Promoter Regions, Genetic , Animals , Antigens, CD34/genetics , Bone Marrow/immunology , Bone Marrow Transplantation , Disease Models, Animal , Dogs , Humans , Leukocyte-Adhesion Deficiency Syndrome/genetics , Leukocyte-Adhesion Deficiency Syndrome/veterinary , Neutrophils/immunology , Transduction, GeneticSubject(s)
DNA Mutational Analysis/methods , Electrophoresis, Polyacrylamide Gel/methods , Integrin beta3/genetics , Platelet Membrane Glycoprotein IIb/genetics , Point Mutation , Polymerase Chain Reaction/methods , Adolescent , Adult , Child, Preschool , Female , Humans , Male , Platelet Glycoprotein GPIIb-IIIa Complex/genetics , Reproducibility of Results , Sensitivity and Specificity , Thrombasthenia/geneticsABSTRACT
BACKGROUND: Glanzmann thrombasthenia (GT) is a congenital bleeding disorder caused by either a lack or dysfunction of the platelet integrin alphaIIbbeta3. OBJECTIVES: To determine the molecular basis of GT in patients from southern India. PATIENTS: Fifteen unrelated patients whose diagnosis was consistent with GT were evaluated. RESULTS: Platelet surface expression of alphaIIbbeta3 was < 10%, 10%-50%, and > 50% of controls in five, nine, and one patient(s), respectively. Immunoblotting of the platelet lysates showed no alphaIIb in 14 patients, and no beta3 in 10 patients, although severely reduced in four patients. Platelet fibrinogen was undetectable in 13 patients, and severely reduced in one patient. One patient showed normal surface alphaIIbbeta3 expression, and normal alphaIIb, beta3 and fibrinogen levels in the lysate. Ten novel candidate disease-causing mutations were identified in 11 patients. The missense mutations included Gly128Ser, Ser287Leu, Gly357Ser, Arg520Trp, Leu799Arg in alphaIIb, and Cys575Gly in beta3. We have already shown that Gly128Ser, Ser287Leu, and Gly357Ser mutations variably affect alphaIIbbeta3 surface expression. The Cys575Gly mutation may disrupt the disulphide link with Cys586 to cause the GT phenotype. The molecular pathology of the other missense mutations is not clear. Two nonsense mutations, Trp-16Stop and Glu715Stop in alphaIIb, and a 7-bp deletion (330-336TCCCCAG) in beta3 are predicted to result in truncated proteins. An IVS15(-1)G --> A mutation in alphaIIb induced a cryptic splice site as confirmed by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Thirteen polymorphisms were also identified (five in alphaIIb and eight in beta3), among which five were novel. CONCLUSIONS: While identifying a significant number of novel mutations causing GT, this study confirms the genetic heterogeneity of the disorder in southern India.
Subject(s)
Blood Platelets/metabolism , Mutation , Platelet Glycoprotein GPIIb-IIIa Complex/genetics , Polymorphism, Genetic , Thrombasthenia/diagnosis , Thrombasthenia/genetics , Adolescent , Adult , Binding Sites , Cell Membrane/metabolism , Child , DNA Mutational Analysis , Female , Fibrinogen/biosynthesis , Humans , Male , Polymorphism, Restriction Fragment LengthABSTRACT
BACKGROUND: Glanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder characterized by lack of platelet aggregation in response to most physiological agonists and caused by either a lack or dysfunction of the platelet integrin alpha(IIb)beta3 (glycoprotein IIb/IIIa). OBJECTIVES: To determine the molecular basis of GT and characterize the mutations by in vitro expression studies. PATIENTS: We studied three unrelated patients from southern India whose diagnosis was consistent with GT. RESULTS: Immunoprecipitation of the cell lysates and immunoblotting showed no detectable mature alpha(IIb) in the G128S mutant, in contrast to 6% and 33% of the normal amount of mature alpha(IIb) in the S287L and G357S mutants, respectively. Pulse-chase analysis demonstrated pro-alpha(IIb) in the mutants comparable with the normal pro-alpha(IIb), but no conversion to mature alpha(IIb) in the G128S mutant, and only trace conversion to mature alpha(IIb) in the S287L and G357S mutants. The disappearance of pro-alpha(IIb) in the three mutants was similar to that in cells expressing normal alpha(IIb)beta3 or alpha(IIb) only. All three mutants demonstrated pro-alpha(IIb)beta3 complexes and co-localized with an ER marker by immunofluorescence. The G128S mutant showed no co-localization with a Golgi marker, and the other two mutants showed minimal and moderate co-localization with the Golgi marker. CONCLUSIONS: These three beta-propeller mutations do not affect the production of pro-alpha(IIb), its ability to complex with beta3, or its stability, but do cause variable defects in transport of pro-alpha(IIb)beta3 complexes from the endoplasmic reticulum to the Golgi.
