- Email: [email protected]
Ontogeny of GAD, an autoantigen in diabetes
MOLECULAR
ons in the gene
Pennacchio, L.A. et al. (1996) Sdme271,l731-1734
down new epilepsy genes Allen, K.M. and Walsh, C. (1996) Nat.
2,516-518
Although many diseases have succumbed to the techniques of molecular biolo:]y, the field of epilepsy has proved difficult. Thus far, a number of heritable epilepsy syndromes have been mapped to specific chromosomal locations, but only a few have been linked to mutations in specific genes: a tRNAmutation in myoclonic epilepsy with ragged red fibers (MERRF), mutations in the nicotinic acetylcholine receptor (~4 in autosomaldominant nocturnal frontal-lobe epilepsy, and a mutation in CL/V3 (a novel gene encoding a 438 amino acid protein of unknown function) in neurcnal ceroid lipofuscinosis. Pennacchio et al. have now identified the gene responsible for a recessively inherited form of progressive myoclonic epilepsy (EPMl, Unverricht-Lundborgtype). They identified a 750 kilobase region on chromosome 21 by linkage diseq!lilibrium and recombination breakpoint mapping in four Finnish pedigrees. cDNA selection revealed that the gene for the cysteine-protease cystatin B was located in this region. Sequencing revealed mutations in the gene (either premature stop codons or alterations in intron-exon boundaries) in three out of four families studied. More importantly, RNA levels for cystatin B were reduced in all four pedigrees. Cystatin B is ubiquitously expressed and is thought to be involved in inactivating proteases that leak out of lysosomes; how a defect in this protein could cause neurological disease is unknown, but it is interesting to speculate that cystatin B might be involved in formation or
maintenance of synaptic vesicles, which have many similarities to lysosomes. In another paper, Allen and Walsh discuss the finding that transgenic mice that are deficient in the proteins involved in presynaptic function or transmitter release are more likely than wild-type mice to have seizures, implying that presynaptic mechanisms (including formation and maintenance of vesicles) may play a pivotal role in epileptogenesis. Exploring the role of cystatin B could therefore provide important clues for the treatment of both heritable and acquired epilepsy. Joy Snider MD, PhD Instructorin Neurology,Departmentof Neurologyand NeurologicalSurgery,CSNSI, WashingtonUniversity School of Medicine,Box 8111,660 S. Euclid Avenue, StLouis,MO68110-1093,USA.
Ontogeny and tissue distribution of Mally, M.I. eta/. (1996) Diabetes 45,496-501 Type I diabetes is an organ-specific autoimmune disease characterized by lymphocytic infiltration of the islets of the pancreas and production of autoantibodies against islet cells. Autoantibodies to a 64 kDa protein can be detected at, and for several years before, the onsd of this disease; this protein is an enzyme, glutamic acid decarboxylase (GAD), which catalyses the synthesis of y-amino butyric acid (GABA). It is present in the brain and, importantly, in pancreatic islets. Other proteins present in the islet cells are also implicated as putative autoantigens in type I diabetes; they include insulin, carboxypeptidase H, heat shock protein 65 (hsp65) and islet cell antigen 69 (ICA69).
MEDICINE
TODAY.
AUGUST
I‘996
rected to GAD65 The biological significance of either GAD or its catalytic product. GA pancreatic islet is still not known. In this paper, Mally and co-workers report the transcriptional expression of both genes for GAD during human pancreatic ontogeny, and also explore the cellular distribution of both GAD proteins in fetal and adult islets. The authors confirmed the previous observations of pan-islet distribution of GAD65 in human pancreas. Th different subsets of differentiate tiated islet cells in the fetal pancreas with varyi levels of GAD65 protein present, demonstrating the plasticity of the islet progenitors. As expected, the synthesis of GAD65 in the pancreas was confined to islets and there was a subset of epithelial cells in the fetal pancreas that showed immunoreactivity to both glucagon and insulin, together with GAD65. The co-expression of glucagon and insulin in fetal pancreatic epithelial cells could indicate a transitional differentiation state; the synthesis of GAD65 in these cells shows that this protein is present in undifferentiated epithelial cells, the most likely precursors of endocrine cells. GAD65 was also found in ductal epithelial cells of the pancreas that lacked either insulin or glucagon. This report also showed that tissues other than pancreas and brain (kidney, adrenal gland and ventricle) contain mRNA for GAD65. The known autoantigens in organ-specific autoimmune diseases other than diabetes are tissue specific and functionally significant.The panislet distribution of GAD65, and also its presence in non-islet tissues, question the role of GAD as a primary autoantigen in type I diabetes, because only p cells of the islet are affected in this autoimmune disease. The reason for the specific vulnerability of p cells resulting in their autoimmune destruction therefore remains a mystery. This study could have been even more informative if the tissue distribution of GAD protein had been included (using immunohistochemistty,
Copyright 01996 Elsevier Science Ltd. All rights reserved. 1357 - 431QP6/!§15.00
rtment of Immunology, 310,200 I ststreetSW,
Ja
Scientist, Department of Genetics, Hospital for Sick Children, 555 University Avenue, Toronto, Canada M56 1X8.
Sickle cell anemia srnd p-thalassemia are caused by a coding defect in the by reduction of its expre been attractive candidates for somatic gene therapy. Since the p-globin gene is small and a well-characterized locus control regulated region (LC , it should be a ‘simple’matter to produce retrovirus vectors for this purpose. After more than ten years of attempts by many labs to transfer p-globin genes, frustrated by sequences that are incompatible with virus replication, a new approach promises high-efficiency transfer of a regulated y-globin gene. Ren and collaborators avoided the recombinogenic sequences present in the p-globin intron 2 and LCR by replacing them with more stable sequences from the a-globin LCF! and the y-globin gene. The retrovirus used by them is based on the N2A retrovirus backbone in which a neo gene is transcribed from the Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR). The human y-globin gene, complete with introns, is cloned in the antisense orientation, and a double copy of the a-globin LCW present in the viral LTRs serves to boost r_globin expression. High viral titers (well above lo6 ml-‘) were obtainerr and high levels of y-globin gene Copyright 01996 Elsevier Science Ltd. All rights reserved
intensive chemotherapy are equally effective in improving disease-free survival in children with AML although I suspect that, for many patients
and their parents, intensive chemotherapy would be a more acceptable option.
OI12Marrow Transpla 50x 234, Depaitment of Haematology, Addenbrooke’s NHS Trust, Cambridge, UK CB2 2QQ.
Acute myeloid leukemia (ANIL) accounts for 15-20% of childhood leukemia. No predisposing factors are known for the majority of children with AML but factors that are associated with an increased incidence include: Down’s syndrome, Fanconi’s anaemia, ionizing radiation, cetiin drugs such as benzene and epipodophyllotoxins, and the myelodysplastic syndromes. Although chemotherapy can induce remissions in W-85% of children with AML, only 3045% of I357 - 43 10/96/$ IS.00
ons in the gene
Pennacchio, L.A. et al. (1996) Sdme271,l731-1734
down new epilepsy genes Allen, K.M. and Walsh, C. (1996) Nat.
2,516-518
Although many diseases have succumbed to the techniques of molecular biolo:]y, the field of epilepsy has proved difficult. Thus far, a number of heritable epilepsy syndromes have been mapped to specific chromosomal locations, but only a few have been linked to mutations in specific genes: a tRNAmutation in myoclonic epilepsy with ragged red fibers (MERRF), mutations in the nicotinic acetylcholine receptor (~4 in autosomaldominant nocturnal frontal-lobe epilepsy, and a mutation in CL/V3 (a novel gene encoding a 438 amino acid protein of unknown function) in neurcnal ceroid lipofuscinosis. Pennacchio et al. have now identified the gene responsible for a recessively inherited form of progressive myoclonic epilepsy (EPMl, Unverricht-Lundborgtype). They identified a 750 kilobase region on chromosome 21 by linkage diseq!lilibrium and recombination breakpoint mapping in four Finnish pedigrees. cDNA selection revealed that the gene for the cysteine-protease cystatin B was located in this region. Sequencing revealed mutations in the gene (either premature stop codons or alterations in intron-exon boundaries) in three out of four families studied. More importantly, RNA levels for cystatin B were reduced in all four pedigrees. Cystatin B is ubiquitously expressed and is thought to be involved in inactivating proteases that leak out of lysosomes; how a defect in this protein could cause neurological disease is unknown, but it is interesting to speculate that cystatin B might be involved in formation or
maintenance of synaptic vesicles, which have many similarities to lysosomes. In another paper, Allen and Walsh discuss the finding that transgenic mice that are deficient in the proteins involved in presynaptic function or transmitter release are more likely than wild-type mice to have seizures, implying that presynaptic mechanisms (including formation and maintenance of vesicles) may play a pivotal role in epileptogenesis. Exploring the role of cystatin B could therefore provide important clues for the treatment of both heritable and acquired epilepsy. Joy Snider MD, PhD Instructorin Neurology,Departmentof Neurologyand NeurologicalSurgery,CSNSI, WashingtonUniversity School of Medicine,Box 8111,660 S. Euclid Avenue, StLouis,MO68110-1093,USA.
Ontogeny and tissue distribution of Mally, M.I. eta/. (1996) Diabetes 45,496-501 Type I diabetes is an organ-specific autoimmune disease characterized by lymphocytic infiltration of the islets of the pancreas and production of autoantibodies against islet cells. Autoantibodies to a 64 kDa protein can be detected at, and for several years before, the onsd of this disease; this protein is an enzyme, glutamic acid decarboxylase (GAD), which catalyses the synthesis of y-amino butyric acid (GABA). It is present in the brain and, importantly, in pancreatic islets. Other proteins present in the islet cells are also implicated as putative autoantigens in type I diabetes; they include insulin, carboxypeptidase H, heat shock protein 65 (hsp65) and islet cell antigen 69 (ICA69).
MEDICINE
TODAY.
AUGUST
I‘996
rected to GAD65 The biological significance of either GAD or its catalytic product. GA pancreatic islet is still not known. In this paper, Mally and co-workers report the transcriptional expression of both genes for GAD during human pancreatic ontogeny, and also explore the cellular distribution of both GAD proteins in fetal and adult islets. The authors confirmed the previous observations of pan-islet distribution of GAD65 in human pancreas. Th different subsets of differentiate tiated islet cells in the fetal pancreas with varyi levels of GAD65 protein present, demonstrating the plasticity of the islet progenitors. As expected, the synthesis of GAD65 in the pancreas was confined to islets and there was a subset of epithelial cells in the fetal pancreas that showed immunoreactivity to both glucagon and insulin, together with GAD65. The co-expression of glucagon and insulin in fetal pancreatic epithelial cells could indicate a transitional differentiation state; the synthesis of GAD65 in these cells shows that this protein is present in undifferentiated epithelial cells, the most likely precursors of endocrine cells. GAD65 was also found in ductal epithelial cells of the pancreas that lacked either insulin or glucagon. This report also showed that tissues other than pancreas and brain (kidney, adrenal gland and ventricle) contain mRNA for GAD65. The known autoantigens in organ-specific autoimmune diseases other than diabetes are tissue specific and functionally significant.The panislet distribution of GAD65, and also its presence in non-islet tissues, question the role of GAD as a primary autoantigen in type I diabetes, because only p cells of the islet are affected in this autoimmune disease. The reason for the specific vulnerability of p cells resulting in their autoimmune destruction therefore remains a mystery. This study could have been even more informative if the tissue distribution of GAD protein had been included (using immunohistochemistty,
Copyright 01996 Elsevier Science Ltd. All rights reserved. 1357 - 431QP6/!§15.00
rtment of Immunology, 310,200 I ststreetSW,
Ja
Scientist, Department of Genetics, Hospital for Sick Children, 555 University Avenue, Toronto, Canada M56 1X8.
Sickle cell anemia srnd p-thalassemia are caused by a coding defect in the by reduction of its expre been attractive candidates for somatic gene therapy. Since the p-globin gene is small and a well-characterized locus control regulated region (LC , it should be a ‘simple’matter to produce retrovirus vectors for this purpose. After more than ten years of attempts by many labs to transfer p-globin genes, frustrated by sequences that are incompatible with virus replication, a new approach promises high-efficiency transfer of a regulated y-globin gene. Ren and collaborators avoided the recombinogenic sequences present in the p-globin intron 2 and LCR by replacing them with more stable sequences from the a-globin LCF! and the y-globin gene. The retrovirus used by them is based on the N2A retrovirus backbone in which a neo gene is transcribed from the Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR). The human y-globin gene, complete with introns, is cloned in the antisense orientation, and a double copy of the a-globin LCW present in the viral LTRs serves to boost r_globin expression. High viral titers (well above lo6 ml-‘) were obtainerr and high levels of y-globin gene Copyright 01996 Elsevier Science Ltd. All rights reserved
intensive chemotherapy are equally effective in improving disease-free survival in children with AML although I suspect that, for many patients
and their parents, intensive chemotherapy would be a more acceptable option.
OI12Marrow Transpla 50x 234, Depaitment of Haematology, Addenbrooke’s NHS Trust, Cambridge, UK CB2 2QQ.
Acute myeloid leukemia (ANIL) accounts for 15-20% of childhood leukemia. No predisposing factors are known for the majority of children with AML but factors that are associated with an increased incidence include: Down’s syndrome, Fanconi’s anaemia, ionizing radiation, cetiin drugs such as benzene and epipodophyllotoxins, and the myelodysplastic syndromes. Although chemotherapy can induce remissions in W-85% of children with AML, only 3045% of I357 - 43 10/96/$ IS.00