The Experts below are selected from a list of 318 Experts worldwide ranked by ideXlab platform
Daisuke Tsuji - One of the best experts on this subject based on the ideXlab platform.
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molecular pathogenesis and therapeutic approach of gm2 gangliosidosis
Yakugaku Zasshi-journal of The Pharmaceutical Society of Japan, 2013Co-Authors: Daisuke TsujiAbstract:Tay-Sachs and Sandhoff diseases (GM2 gangliosidoses) are autosomal recessive lysosomal storage diseases caused by gene mutations in HEXA and HEXB, each encoding human lysosomal β-hexosaminidase α-subunits and β-subunits, respectively. In Tay-Sachs disease, excessive accumulation of GM2 ganglioside (GM2), mainly in the central nervous system, is caused by a deficiency of the HEXA isozyme (αβ heterodimer), resulting in progressive neurologic disorders. In Sandhoff disease, combined deficiencies of HEXA and HexB (ββ homodimer) cause not only the accumulation of GM2 but also of oligosaccharides carrying terminal N-acetylhexosamine residues (GlcNAc-oligosaccharides), resulting in systemic manifestations including hepatosplenomegaly as well as neurologic symptoms. Hence there is little clinically effective treatment for these GM2 gangliosidoses. Recent studies on the molecular pathogenesis in Sandhoff disease patients and disease model mice have shown the involvement of microglial activation and chemokine induction in neuroinflammation and neurodegeneration in this disease. Experimental and therapeutic approaches, including recombinant enzyme replacement, have been performed using Sandhoff disease model mice, suggesting the future application of novel techniques to treat GM2 gangliosidoses (Hex deficiencies), including Sandhoff disease as well as Tay-Sachs disease. In this study, we isolated astrocytes and microglia from the neonatal brain of Sandhoff disease model mice and demonstrated abnormalities of glial cells. Moreover, we demonstrated the therapeutic effect of an intracerebroventricular administration of novel recombinant human HEXA carrying a high content of M6P residue in Sandhoff disease model mice.
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therapeutic potential of intracerebroventricular replacement of modified human β hexosaminidase b for gm2 gangliosidosis
Molecular Therapy, 2011Co-Authors: Kazuhiko Matsuoka, Keisuke Kitakaze, Daisuke Tsuji, Hitoshi Sakuraba, Tomomi Tamura, Yukie Dohzono, Kazuki Ohno, Seiji Saito, Kohji ItohAbstract:To develop a novel enzyme replacement therapy for neurodegenerative Tay-Sachs disease (TSD) and Sandhoff disease (SD), which are caused by deficiency of β-hexosaminidase (Hex) A, we designed a genetically engineered HEXB encoding the chimeric human β-subunit containing partial amino acid sequence of the α-subunit by structure-based homology modeling. We succeeded in producing the modified HexB by a Chinese hamster ovary (CHO) cell line stably expressing the chimeric HEXB, which can degrade artificial anionic substrates and GM2 ganglioside in vitro, and also retain the wild-type (WT) HexB-like thermostability in the presence of plasma. The modified HexB was efficiently incorporated via cation-independent mannose 6-phosphate receptor into fibroblasts derived from Tay-Sachs patients, and reduced the GM2 ganglioside accumulated in the cultured cells. Furthermore, intracerebroventricular administration of the modified HexB to Sandhoff mode mice restored the Hex activity in the brains, and reduced the GM2 ganglioside storage in the parenchyma. These results suggest that the intracerebroventricular enzyme replacement therapy involving the modified HexB should be more effective for Tay-Sachs and Sandhoff than that utilizing the HEXA, especially as a low-antigenic enzyme replacement therapy for Tay-Sachs patients who have endogenous WT HexB.
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highly phosphomannosylated enzyme replacement therapy for gm2 gangliosidosis
Annals of Neurology, 2011Co-Authors: Daisuke Tsuji, Hiromi Akeboshi, Kazuhiko Matsuoka, Hiroko Yasuoka, Eri Miyasaki, Yoshiko Kasahara, Ikuo Kawashima, Yasunori Chiba, Yoshifumi Jigami, Takao TakiAbstract:Objective: Novel recombinant human lysosomal β-hexosaminidase A (HEXA) was developed for enzyme replacement therapy (ERT) for Tay-Sachs and Sandhoff diseases, ie, autosomal recessive GM2 gangliosidoses, caused by HEXA deficiency. Methods: A recombinant human HEXA (Om4HEXA) with a high mannose 6-phosphate (M6P)-type-N-glycan content, which was produced by a methylotrophic yeast strain, Ogataea minuta, overexpressing the OmMNN4 gene, was intracerebroventricularly (ICV) administered to Sandhoff disease model mice (Hexb−/− mice) at different doses (0.5–2.5mg/kg), and then the replacement and therapeutic effects were examined. Results: The Om4HEXA was widely distributed across the ependymal cell layer, dose-dependently restored the enzyme activity due to uptake via cell surface cation-independent M6P receptor (CI-M6PR) on neural cells, and reduced substrates, including GM2 ganglioside (GM2), asialo GM2 (GA2), and oligosaccharides with terminal N-acetylglucosamine residues (GlcNAc-oligosaccharides), accumulated in brain parenchyma. A significant inhibition of chemokine macrophage inflammatory protein-1 α (MIP-1α) induction was also revealed, especially in the hindbrain (<63%). The decrease in central neural storage correlated with an improvement of motor dysfunction as well as prolongation of the lifespan. Interpretation: This lysosome-directed recombinant human enzyme drug derived from methylotrophic yeast has the high therapeutic potential to improve the motor dysfunction and quality of life of the lysosomal storage diseases (LSDs) patients with neurological manifestations. We emphasize the importance of neural cell surface M6P receptor as a delivery target of neural cell-directed enzyme replacement therapy (NCDERT) for neurodegenerative metabolic diseases. ANN NEUROL 2010
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Introduction of an N-Glycan Sequon Into HEXA Enhances Human β-Hexosaminidase Cellular Uptake in a Model of Sandhoff Disease
Molecular therapy : the journal of the American Society of Gene Therapy, 2010Co-Authors: Kazuhiko Matsuoka, Daisuke Tsuji, Hitoshi Sakuraba, Seiichi Aikawa, Fumiko Matsuzawa, Kohji ItohAbstract:Human lysosomal beta-hexosaminidase A is a heterodimer composed of alpha- and beta-subunits encoded by HEXA and HEXB, respectively. We genetically introduced an additional N-glycosylation sequon into HEXA, which caused amino acid substitutions (S51 to N and A53 to T) at homologous positions to N84 and T86 in the beta-subunit. The mutant HEXA (NgHEXA) obtained from a Chinese hamster ovary (CHO) cell line co-expressing the mutated HEXA and wild-type HEXB complementary DNAs was demonstrated to contain an additional mannose-6-phosphate (M6P)-type-N-glycan. NgHEXA was more efficiently taken up than the wild-type HEXA and delivered to lysosomes, where it degraded accumulated substrates including GM2 ganglioside (GM2) when administered to cultured fibroblasts derived from a Sandhoff disease (SD) patient. On intracerebroventricular (i.c.v.) administration of NgHEXA to SD model mice, NgHEXA more efficiently restored the HEXA activity and reduced the GM2 and GA2 (asialoGM2) accumulated in neural cells of the brain parenchyma than the wild-type HEXA. These findings indicate that i.c.v. administration of the modified human HEXA with an additional M6P-type N-glycan is applicable for enzyme replacement therapy (ERT) involving an M6P-receptor as a molecular target for HEXA deficiencies including Tay-Sachs disease and SD.
Sohita Dhillon - One of the best experts on this subject based on the ideXlab platform.
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Spotlight on DTPa-HBV-IPV/Hib Vaccine (Infanrix HEXA™)†
BioDrugs, 2010Co-Authors: Sohita DhillonAbstract:Infanrix HEXA™, administered intramuscularly, is a diphtheria, tetanus, acellular pertussis, hepatitis B (HBV), inactivated poliomyelitis and Haemophilus influenzae type b (Hib) conjugate vaccine, indicated for primary and booster vaccination of infants. Infanrix HEXA™ should be administered as a two- or three-dose primary vaccination course in infants aged ≤6 months, followed by booster vaccination between 11 and 18 months of age, with an interval of at least 6 months between the last dose of primary vaccination and the booster dose. This spotlight reviews the immunogenicity and protective effectiveness, as well as the reactogenicity and safety of Infanrix HEXA™. Infanrix HEXA™ as primary and booster vaccination was safe and highly immunogenic for all its component toxoids/antigens in infants aged
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Spotlight on DTPa-HBV-IPV/Hib Vaccine (Infanrix HEXA™)†
BioDrugs : clinical immunotherapeutics biopharmaceuticals and gene therapy, 2010Co-Authors: Sohita DhillonAbstract:Infanrix HEXA™, administered intramuscularly, is a diphtheria, tetanus, acellular pertussis, hepatitis B (HBV), inactivated poliomyelitis and Haemophilus influenzae type b (Hib) conjugate vaccine, indicated for primary and booster vaccination of infants. Infanrix HEXA™ should be administered as a two- or three-dose primary vaccination course in infants aged ≤6 months, followed by booster vaccination between 11 and 18 months of age, with an interval of at least 6 months between the last dose of primary vaccination and the booster dose. This spotlight reviews the immunogenicity and protective effectiveness, as well as the reactogenicity and safety of Infanrix HEXA™.
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DTPa-HBV-IPV/Hib Vaccine (Infanrix HEXA): A Review of its Use as Primary and Booster Vaccination.
Drugs, 2010Co-Authors: Sohita DhillonAbstract:Infanrix HEXA™, administered intramuscularly, is a diphtheria, tetanus, acellular pertussis, hepatitis B (HBV), inactivated poliomyelitis and Haemophilus influenzae type b (Hib) conjugate vaccine, indicated for primary and booster vaccination of infants. Infanrix HEXA™ should be administered as a two- or three-dose primary vaccination course in infants aged ≤6 months, followed by booster vaccination between 11 and 18 months of age, with an interval of at least 6 months between the last dose of primary vaccination and the booster dose. This article reviews the immunogenicity and protective effectiveness, as well as the reactogenicity and safety of Infanrix HEXA™. Infanrix HEXA™ as primary and booster vaccination was safe and highly immunogenic for all its component toxoids/antigens in infants aged
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DTPa-HBV-IPV/Hib Vaccine (Infanrix HEXA™)
Drugs, 2010Co-Authors: Sohita DhillonAbstract:Infanrix HEXA™, administered intramuscularly, is a diphtheria, tetanus, acellular pertussis, hepatitis B (HBV), inactivated poliomyelitis and Haemophilus influenzae type b (Hib) conjugate vaccine, indicated for primary and booster vaccination of infants. Infanrix HEXA™ should be administered as a two- or three-dose primary vaccination course in infants aged ≤6 months, followed by booster vaccination between 11 and 18 months of age, with an interval of at least 6 months between the last dose of primary vaccination and the booster dose. This article reviews the immunogenicity and protective effectiveness, as well as the reactogenicity and safety of Infanrix HEXA™. Infanrix HEXA™ as primary and booster vaccination was safe and highly immunogenic for all its component toxoids/antigens in infants aged
Angela Gritti - One of the best experts on this subject based on the ideXlab platform.
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Novel bicistronic lentiviral vectors correct β-Hexosaminidase deficiency in neural and hematopoietic stem cells and progeny: implications for in vivo and ex vivo gene therapy of GM2 gangliosidosis.
Neurobiology of disease, 2019Co-Authors: Francesca Ornaghi, Francesco Morena, Martina Bazzucchi, Sabata Martino, Davide Sala, Fabiana Tedeschi, Maria Chiara Maffia, Manuela Valsecchi, Massimo Aureli, Angela GrittiAbstract:The favorable outcome of in vivo and ex vivo gene therapy approaches in several Lysosomal Storage Diseases suggests that these treatment strategies might equally benefit GM2 gangliosidosis. Tay-Sachs and Sandhoff disease (the main forms of GM2 gangliosidosis) result from mutations in either the HEXA or HEXB genes encoding, respectively, the α- or β-subunits of the lysosomal β-Hexosaminidase enzyme. In physiological conditions, α- and β-subunits combine to generate β-Hexosaminidase A (HEXA, αβ) and β-Hexosaminidase B (HexB, ββ). A major impairment to establishing in vivo or ex vivo gene therapy for GM2 gangliosidosis is the need to synthesize the α- and β-subunits at high levels and with the correct stoichiometric ratio, and to safely deliver the therapeutic products to all affected tissues/organs. Here, we report the generation and in vitro validation of novel bicistronic lentiviral vectors (LVs) encoding for both the murine and human codon optimized HEXA and Hexb genes. We show that these LVs drive the safe and coordinate expression of the α- and β-subunits, leading to supranormal levels of β-Hexosaminidase activity with prevalent formation of a functional HEXA in SD murine neurons and glia, murine bone marrow-derived hematopoietic stem/progenitor cells (HSPCs), and human SD fibroblasts. The restoration/overexpression of β-Hexosaminidase leads to the reduction of intracellular GM2 ganglioside storage in transduced and in cross-corrected SD murine neural progeny, indicating that the transgenic enzyme is secreted and functional. Importantly, bicistronic LVs safely and efficiently transduce human neurons/glia and CD34+ HSPCs, which are target and effector cells, respectively, in prospective in vivo and ex vivo GT approaches. We anticipate that these bicistronic LVs may overcome the current requirement of two vectors co-delivering the α- or β-subunits genes. Careful assessment of the safety and therapeutic potential of these bicistronic LVs in the SD murine model will pave the way to the clinical development of LV-based gene therapy for GM2 gangliosidosis.
Richard L. Proia - One of the best experts on this subject based on the ideXlab platform.
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distribution of enzyme bearing cells in gm2 gangliosidosis mice regionally specific pattern of cellular infiltration following bone marrow transplantation
Acta Neuropathologica, 2000Co-Authors: Yasushi Oya, Francine Norflus, Richard L. Proia, Cynthia J Tifft, Clarita Langaman, Kinuko SuzukiAbstract:Tissue distribution of β-hexosaminidase was investigated using 5-bromo-4-chloro-3-indolyl N-acetyl β-d-glucosaminide (X-Hex) as substrate in wild-type mice, four GM2 gangliosidosis model mice (HEXA-/-, Hexb-/-, Gm2a-/- and HEXA-/-Hexb-/-) and Hexb-/- mice that received bone marrow transplantation (BMT). In wild-type mice histochemical localization of β-hexosaminidase was detected in the perikarya of the majority of neurons, small process-bearing microglial cells, perivascular macrophages, and macrophages in the choroid plexus and leptomeninges. X-Hex positivity was also noted in the renal tubular epithelium and macrophages in the liver and spleen. The staining pattern in the Gm2a-/- and HEXA-/- mice was generally similar to those of wild type, but in these mice, X-Hex stain was also noted in some storage neurons with swollen perikarya. No X-Hex-positive cells were detected in Hexb-/- or HEXA-/-Hexb-/- (DKO) mice. In Hexb-/- mice that received wild-type BMT (Hexb-/-+WBMT), many X-Hex-positive cells were detected in the spleen, and to a far lesser extent, in liver and kidney. In the CNS of these mice, X-Hex-positive cells were largely detected in the leptomeninges and choroid plexus. Some positive cells were also detected, mostly in the perivascular regions of the cerebrum, in particular in the regions of the posterior thalamus, brain stem and spinal cord. Some of X-Hex-positive cells were immunoreactive with Mac-1 and F4/80 antibodies and, thus, were cells of microglia/macrophage lineage. X-Hex-positive staining was not detected in neurons in these mice despite clinical improvement following BMT. This is the first time, as far as we know, that the regional distribution of the donor cells in the CNS has been investigated in a model of neuronal storage disease. Our study indicated that donor-derived cells of microglia/macrophage lineage infiltrated the CNS in a regionally specific manner following the BMT.
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Promoters for the human beta-hexosaminidase genes, HEXA and HEXB.
DNA and cell biology, 1996Co-Authors: Francine Norflus, Shoji Yamanaka, Richard L. ProiaAbstract:Human lysosomal beta-hexosaminidases are encoded by two genes, HEXA and HEXB, specifying an alpha- and a beta-subunit, respectively. The subunits dimerize to form beta-hexosaminidase A (alpha beta), beta-hexosaminidase B (beta beta), and beta-hexosaminidase S (alpha alpha). This enzyme system has the capacity to degrade a variety of cellular substrates: oligosaccharides, glycosaminoglycans, and glycolipids containing beta-linked N-acetylglucosaminyl or N-galactosaminyl residues. Mutations in either the HEXA gene or HEXB gene lead to an accumulation of GM2 ganglioside in neurons, resulting in the severe neurodegenerative disorders termed the GM2 gangliosidoses. To identify the DNA elements responsible for hexosaminidase expression, we ligated the 5'-flanking sequences of both the human and mouse hexosaminidase genes to a chloramphenicol acetyltransferase (CAT) gene. The resulting plasmids were transfected into NIH-3T3 cells and CAT activity was determined as a measure of promoter strength. By 5' deletion analysis, it was found that essential sequences for HEXA expression resided within a 40-bp region between 100 bp and 60 bp upstream of the ATG initiation codon. This area contained two potential estrogen response element half-sites as well as potential binding sites for transcription factors NF-E1 and AP-2. Similarly, important HEXB promoter sequences were localized to a 60-bp region between 150 bp and 90 bp upstream of the ATG codon. By performing scanning mutagenesis on a 60-bp region within the 150-bp HEXB construct, we defined an essential promoter element of 12 bp that contained two potential AP-1 sites. The mouse HEXA and Hexb 5'-flanking sequences were found to contain regions similar in sequence, location, and activity to the essential promoter elements defined in the cognate human genes. No sequence similarity was found, however, between 5'-flanking regions of the HEXA and HEXB genes. These essential promoter elements represent potential sites for HEXA and HEXB mutations that could alter enzyme expression in Tay-Sachs and Sandhoff diseases, respectively.
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The molecular basis of HEXA mRNA deficiency caused by the most common Tay-Sachs disease mutation.
American journal of human genetics, 1995Co-Authors: Debra J. Boles, Richard L. ProiaAbstract:Tay-Sachs disease (TSD) is a catastrophic neurodegenerative disorder caused by mutations in the HEXA gene. The most common TSD allele worldwide contains a 4-bp insertion in exon 11 that produces a downstream premature termination codon. Despite normal transcription of this allele, HEXA mRNA is severely reduced, indicating that the HEXA transcript must be unstable. Minigenes of HEXA were constructed and expressed in mouse L cells, to investigate the relationship between the 4-bp insertion and mRNA deficiency. We conclude that the mRNA instability is caused by the premature termination codon and not by a cryptic mutation or by the 4-bp insertion directly and that degradation occurs coincident with or after splicing.
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Structure and Expression of the Mouse β-Hexosaminidase Genes, HEXA and Hexb
Genomics, 1994Co-Authors: Shoji Yamanaka, Olivia N. Johnson, Francine Norflus, Debra J. Boles, Richard L. ProiaAbstract:Two genes, HEXA and HEXB, encode the alpha- and beta-subunits, respectively, of human beta-hexosaminidase. In the mouse, the corresponding genes are termed HEXA and Hexb. The subunits dimerize to yield three isozymes, beta-hexosaminidase A (alpha beta), B (beta beta), and S (alpha alpha), that have the capacity to degrade a variety of substrates containing beta-linked N-acetylglucosamine and N-acetylgalactosamine residues. Mutations in the HEXA or HEXB gene resulting in a beta-hexosaminidase deficiency cause Tay-Sachs or Sandhoff disease, respectively. As a prelude to the creation of mouse models of these lysosomal storage diseases, we have characterized the molecular biology of the mouse beta-hexosaminidase system. Protein sequences derived from the cloned HEXA and Hexb cDNAs were 55% identical to each other and were also very similar to the cognate human sequences: 84% sequence identity with human HEXA and 75% with HEXB. The mouse hexosaminidase subunits, when expressed in HeLa cells from the cDNAs, displayed specificity toward synthetic substrates similar to the human subunits. The HEXA and Hexb genes were 25 and 22 kb in length, respectively. Each gene was divided into 14 exons, with the positions of introns precisely matching those of the corresponding human genes. The 5' flanking regions of the mouse genes demonstrated promoter activity as ascertained by their ability to drive chloramphenicol acetyltransferase gene expression in transfected NIH 3T3 cells. The sequences of these regulatory regions were G+C-rich in the 200 bp upstream of the respective initiator ATGs. Several putative promoter elements were present, including Sp1, AP2, CAAT, and TATA motifs.(ABSTRACT TRUNCATED AT 250 WORDS)
Dennis P. O'brien - One of the best experts on this subject based on the ideXlab platform.
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GM2 Gangliosidosis in Shiba Inu Dogs with an In-Frame Deletion in HEXB.
Journal of veterinary internal medicine, 2017Co-Authors: A. Kolicheski, David A. Wenger, Gary S. Johnson, N. A. Villani, Dennis P. O'brien, Tendai Mhlanga-mutangadura, K. Mikoloski, J.s. Eagleson, Jeremy F. Taylor, Robert D. SchnabelAbstract:Consistent with a tentative diagnosis of neuronal ceroid lipofuscinosis (NCL), autofluorescent cytoplasmic storage bodies were found in neurons from the brains of 2 related Shiba Inu dogs with a young-adult onset, progressive neurodegenerative disease. Unexpectedly, no potentially causal NCL-related variants were identified in a whole-genome sequence generated with DNA from 1 of the affected dogs. Instead, the whole-genome sequence contained a homozygous 3 base pair (bp) deletion in a coding region of HEXB. The other affected dog also was homozygous for this 3-bp deletion. Mutations in the human HEXB ortholog cause Sandhoff disease, a type of GM2 gangliosidosis. Thin-layer chromatography confirmed that GM2 ganglioside had accumulated in an affected Shiba Inu brain. Enzymatic analysis confirmed that the GM2 gangliosidosis resulted from a deficiency in the HEXB encoded protein and not from a deficiency in products from HEXA or GM2A, which are known alternative causes of GM2 gangliosidosis. We conclude that the homozygous 3-bp deletion in HEXB is the likely cause of the Shiba Inu neurodegenerative disease and that whole-genome sequencing can lead to the early identification of potentially disease-causing DNA variants thereby refocusing subsequent diagnostic analyses toward confirming or refuting candidate variant causality.
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GM2 gangliosidosis associated with a HEXA missense mutation in Japanese Chin dogs: a potential model for Tay Sachs disease
Molecular genetics and metabolism, 2012Co-Authors: Douglas N. Sanders, David A. Wenger, Gary S. Johnson, Rong Zeng, Gayle C. Johnson, Jared E. Decker, Martin L. Katz, Simon R. Platt, Dennis P. O'brienAbstract:GM2 gangliosidosis is a fatal lysosomal storage disease caused by a deficiency of β-hexosaminidase (EC 3.2.1.52). There are two major isoforms of the enzyme: hexosaminidase A composed of an α and a β subunit (encoded by HEXA and HEXB genes, respectively); and, hexosaminidase B composed of two β subunits. Hexosaminidase A requires an activator protein encoded by GM2A to catabolize GM2 ganglioside, but even in the absence of the activator protein, it can hydrolyze the synthetic substrates commonly used to assess enzyme activity. GM2 gangliosidosis has been reported in Japanese Chin dogs, and we identified the disease in two related Japanese Chin dogs based on clinical signs, histopathology and elevated brain GM2 gangliosides. As in previous reports, we found normal or elevated hexosaminidase activity when measured with the synthetic substrates. This suggested that the canine disease is analogous to human AB variant of G(M2) gangliosidosis, which results from mutations in GM2A. However, only common neutral single nucleotide polymorphisms were found upon sequence analysis of the canine ortholog of GM2A from the affected Japanese Chins. When the same DNA samples were used to sequence HEXA, we identified a homozygous HEXA:c967G>A transition which predicts a p.E323K substitution. The glutamyl moiety at 323 is known to make an essential contribution to the active site of hexosaminidase A, and none of the 128 normal Japanese Chins and 92 normal dogs of other breeds that we tested was homozygous for HEXA:c967A. Thus it appears that the HEXA:c967G>A transition is responsible for the GM2 gangliosidosis in Japanese Chins.
