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Canine GM2-gangliosidosis (Sandhoff disease) caused by a 3 base pair deletion in the HEXB gene
Abstracts / Molecular Genetics and Metabolism 120 (2016) S17–S145
outcome. (This work is part of our lysosome storage disease research project and quality improvement study. Sponsored by Pfizer/ACMG 2016 Fellowship Award and Resident Research Award from OUHSC Children’s Hospital Research Foundation.)
doi:10.1016/j.ymgme.2016.11.361
353 Different ARSB variants causing mucopolysaccharidosis type VI in dogs Ping Wang, Carol Margolis, Gloria Lin, Karthik Raj, Rachel Han, Lisa Berman, Polly Foureman, Adrian Sewell, Urs Giger, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, United States Mucopolysaccharidoses (MPS) type VI (Maroteaux-Lamy disease, OMIM # 253200) caused by a severe deficiency of arylsulphatase B (ARSB, EC 3.1.6.12) has been reported in human patients and companion animals. MPS VI affected animals (OMIA # 000666) show clinical signs of stunted growth, facial dysmorphia, skeletal deformities, organ enlargements and corneal opacities similar to humans. As part of the NIH National Referral Center for Animal Models of Human Genetic Disease (NIH OD 010939), we describe here the biochemical and molecular characterization of MPS VI in different breeds of dogs. Clinically affected dogs showed many metachromatic granules in leukocytes and a strongly positive urinary mucopolysaccharide spot test due to excessive dermatan sulfate excretion. Tested leukocytes, fibroblasts and/or liver tissue revealed a complete absence of ARSB activity. The ARSB coding and adjacent intronic regions were amplified and sequenced and the results were compared to the CanFam 3.1: CM000003.3 and NCBI RefSeq# NM_001048133.1, and also the sequences from healthy dogs of the same breed. Affected dogs were found to be homozygous for a single base missense (SIFT deleterious) mutation, two polynucleotide deletions, and an early nonsense mutation in Miniature Pinschers, Miniature Schnauzers, Toy Poodles and Great Danes, respectively. Utilizing these breed-specific genomic variants, DNA tests were established to screen related dogs and breeds at the PennGen Laboratories of the School of Veterinary Medicine, University of Pennsylvania. Based upon screening results MPS VI is commonly found in Miniature Pinschers, and like in cats, dogs with MPS VI serve as valuable animal models for studying therapeutic safety and efficacy in human patients.
doi:10.1016/j.ymgme.2016.11.362
354 Canine GM2-gangliosidosis (Sandhoff disease) caused by a 3 base pair deletion in the HEXB gene Ping Wang, Evelyn Galban, Paula S Henthorn, Gloria Lin, Teiko Takedai, Margret Casal, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, United States GM2-gangliosidosis type II (Sandhoff disease, O variant) is a fatal neurodegenerative lysosomal storage disease caused by mutations in the HEXB gene, coding for the beta subunit of β-hexosaminidase type A (HEX-A) and B (HEX-B). Deficiencies in HEX-A and HEX-B lead to accumulation of GM2 gangliosides in neurons and thus to neuronal dysfunction and degeneration. This study characterizes the
S137
phenotype and genotype of a naturally occurring GM2-gangliosidosis in a Shiba Inu dog. A 14-month-old, female Shiba Inu presented with clinical signs of gradually decreasing coordination, difficulty ambulating as well as standing, and tremors. Neurologic evaluation demonstrated the presence of severe cerebellar ataxia, bilateral lack of menace response, and severe intention tremors. Blood smears revealed storage granules in leukocytes, and brain magnetic resonance images were indicative of a neurodegenerative disease. Lysosomal enzymes assays performed on plasma and leukocytes revealed a deficiency in the activities of both HEX-A and HEX-B. Sequencing of the HEXB gene identified a homozygous sequential 3base pair deletion in exon 7 (c.615_617delCCT), which predicts the loss of a leucine residue at amino acid position 206 (p.Leu206del). Protein structure modeling studies predict that the missing leucine could have a deleterious effect on the functional structures of the β subunit of HEX-A and HEX-B. Using this genetic variant as an informative molecular marker, an allelic discrimination real-time PCR based genotyping assay was established to detect the mutant allele. Two hundred clinically healthy dogs of various breeds were homozygous for the wild-type allele, suggesting that this is not a polymorphism in dogs but a unique, disease-causing mutation in the affected dog. This is the first Sandhoff GM2- gangliosidosis described in the Shiba Inu breed and only the second recognized genetic variation responsible for this disorder in dogs.
doi:10.1016/j.ymgme.2016.11.363
355 PRX-102 for treating Fabry disease: immunogenicity and PK results from a phase 1-2 study David Warnocka, Derralynn Hughesb, Simeon Boydc, Pilar Giraldod, Derlis Gonzaleze, Myrl Holidaf, Ozlem Goker-Alpang, Gustavo Maegawah, Mohamed Attai, Kathy Nichollsj,k, Raphael Schiffmannl, Ahmad Tuffaham, Martha Charneyn, Raul Chertkoff o, Sari Alonp, Einat Brill-Almonp, aUAB, Birmingham, AL, United States, bUniversity Colege London, London, United Kingdom, cUC Davis Medical Center, Sacramento, CA, United States, dHospital de Dia Quiron, Zaragoza, Spain, eInstituto Privado De Hematologia Investigacion Clinica, Asuncion, Paraguay, f University of Iowa, Iowa City, IA, United States, gO&O Alpan LLC, Fairfax, VA, United States, hUniversity of Florida, Gainesville, FL, United States, i Johns Hopkins University School of Medicine, Baltimore, MD, United States, j Royal Melbourne Hospital, Melbourne, Australia, kThe University of Melbourne, PArkville, Australia, lBaylor University, Dallas, TX, United States, mKansas University Medical Center, Kansas City, KS, United States, n Pharmacokinetics Consultant, Toronto, ON, Canada, oProtalix, Carmiel, AL, United States, pProtalix, Carmiel, Israel PRX-102 is a novel, chemically modified, α-galactosidase A for the treatment of Fabry disease (FD). Immune responses to therapeutic protein could potentially impact its efficacy and-or safety. In FD, an X-linked disorder caused by the loss of function of the lysosomal enzyme α-galactosidase-A, anti-drug antibody (ADA) formation towards the enzyme has been shown to occur in a high percentage of male patients, especially with nonsense mutations. Immunogenicity and its impact on plasma PRX-102 pharmacokinetics (PK) was evaluated in 16 FD patients participating in a Phase 1/2 study of PRX-102 administered IV every 2 weeks in 3 cohorts (cohort 1, 2 and 3 received 0.2, 1.0 and 2.0 mg/kg PRX-102, respectively). Results: Immunogenicity: Three (3) male patients developed treatment induced IgG antibodies to PRX-102 (ADA +); two patients from Cohort 1 and one from Cohort 2. There were no ADA+ patients in Cohort 3. All 3 ADA+ patients became negative in the second year of
outcome. (This work is part of our lysosome storage disease research project and quality improvement study. Sponsored by Pfizer/ACMG 2016 Fellowship Award and Resident Research Award from OUHSC Children’s Hospital Research Foundation.)
doi:10.1016/j.ymgme.2016.11.361
353 Different ARSB variants causing mucopolysaccharidosis type VI in dogs Ping Wang, Carol Margolis, Gloria Lin, Karthik Raj, Rachel Han, Lisa Berman, Polly Foureman, Adrian Sewell, Urs Giger, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, United States Mucopolysaccharidoses (MPS) type VI (Maroteaux-Lamy disease, OMIM # 253200) caused by a severe deficiency of arylsulphatase B (ARSB, EC 3.1.6.12) has been reported in human patients and companion animals. MPS VI affected animals (OMIA # 000666) show clinical signs of stunted growth, facial dysmorphia, skeletal deformities, organ enlargements and corneal opacities similar to humans. As part of the NIH National Referral Center for Animal Models of Human Genetic Disease (NIH OD 010939), we describe here the biochemical and molecular characterization of MPS VI in different breeds of dogs. Clinically affected dogs showed many metachromatic granules in leukocytes and a strongly positive urinary mucopolysaccharide spot test due to excessive dermatan sulfate excretion. Tested leukocytes, fibroblasts and/or liver tissue revealed a complete absence of ARSB activity. The ARSB coding and adjacent intronic regions were amplified and sequenced and the results were compared to the CanFam 3.1: CM000003.3 and NCBI RefSeq# NM_001048133.1, and also the sequences from healthy dogs of the same breed. Affected dogs were found to be homozygous for a single base missense (SIFT deleterious) mutation, two polynucleotide deletions, and an early nonsense mutation in Miniature Pinschers, Miniature Schnauzers, Toy Poodles and Great Danes, respectively. Utilizing these breed-specific genomic variants, DNA tests were established to screen related dogs and breeds at the PennGen Laboratories of the School of Veterinary Medicine, University of Pennsylvania. Based upon screening results MPS VI is commonly found in Miniature Pinschers, and like in cats, dogs with MPS VI serve as valuable animal models for studying therapeutic safety and efficacy in human patients.
doi:10.1016/j.ymgme.2016.11.362
354 Canine GM2-gangliosidosis (Sandhoff disease) caused by a 3 base pair deletion in the HEXB gene Ping Wang, Evelyn Galban, Paula S Henthorn, Gloria Lin, Teiko Takedai, Margret Casal, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, United States GM2-gangliosidosis type II (Sandhoff disease, O variant) is a fatal neurodegenerative lysosomal storage disease caused by mutations in the HEXB gene, coding for the beta subunit of β-hexosaminidase type A (HEX-A) and B (HEX-B). Deficiencies in HEX-A and HEX-B lead to accumulation of GM2 gangliosides in neurons and thus to neuronal dysfunction and degeneration. This study characterizes the
S137
phenotype and genotype of a naturally occurring GM2-gangliosidosis in a Shiba Inu dog. A 14-month-old, female Shiba Inu presented with clinical signs of gradually decreasing coordination, difficulty ambulating as well as standing, and tremors. Neurologic evaluation demonstrated the presence of severe cerebellar ataxia, bilateral lack of menace response, and severe intention tremors. Blood smears revealed storage granules in leukocytes, and brain magnetic resonance images were indicative of a neurodegenerative disease. Lysosomal enzymes assays performed on plasma and leukocytes revealed a deficiency in the activities of both HEX-A and HEX-B. Sequencing of the HEXB gene identified a homozygous sequential 3base pair deletion in exon 7 (c.615_617delCCT), which predicts the loss of a leucine residue at amino acid position 206 (p.Leu206del). Protein structure modeling studies predict that the missing leucine could have a deleterious effect on the functional structures of the β subunit of HEX-A and HEX-B. Using this genetic variant as an informative molecular marker, an allelic discrimination real-time PCR based genotyping assay was established to detect the mutant allele. Two hundred clinically healthy dogs of various breeds were homozygous for the wild-type allele, suggesting that this is not a polymorphism in dogs but a unique, disease-causing mutation in the affected dog. This is the first Sandhoff GM2- gangliosidosis described in the Shiba Inu breed and only the second recognized genetic variation responsible for this disorder in dogs.
doi:10.1016/j.ymgme.2016.11.363
355 PRX-102 for treating Fabry disease: immunogenicity and PK results from a phase 1-2 study David Warnocka, Derralynn Hughesb, Simeon Boydc, Pilar Giraldod, Derlis Gonzaleze, Myrl Holidaf, Ozlem Goker-Alpang, Gustavo Maegawah, Mohamed Attai, Kathy Nichollsj,k, Raphael Schiffmannl, Ahmad Tuffaham, Martha Charneyn, Raul Chertkoff o, Sari Alonp, Einat Brill-Almonp, aUAB, Birmingham, AL, United States, bUniversity Colege London, London, United Kingdom, cUC Davis Medical Center, Sacramento, CA, United States, dHospital de Dia Quiron, Zaragoza, Spain, eInstituto Privado De Hematologia Investigacion Clinica, Asuncion, Paraguay, f University of Iowa, Iowa City, IA, United States, gO&O Alpan LLC, Fairfax, VA, United States, hUniversity of Florida, Gainesville, FL, United States, i Johns Hopkins University School of Medicine, Baltimore, MD, United States, j Royal Melbourne Hospital, Melbourne, Australia, kThe University of Melbourne, PArkville, Australia, lBaylor University, Dallas, TX, United States, mKansas University Medical Center, Kansas City, KS, United States, n Pharmacokinetics Consultant, Toronto, ON, Canada, oProtalix, Carmiel, AL, United States, pProtalix, Carmiel, Israel PRX-102 is a novel, chemically modified, α-galactosidase A for the treatment of Fabry disease (FD). Immune responses to therapeutic protein could potentially impact its efficacy and-or safety. In FD, an X-linked disorder caused by the loss of function of the lysosomal enzyme α-galactosidase-A, anti-drug antibody (ADA) formation towards the enzyme has been shown to occur in a high percentage of male patients, especially with nonsense mutations. Immunogenicity and its impact on plasma PRX-102 pharmacokinetics (PK) was evaluated in 16 FD patients participating in a Phase 1/2 study of PRX-102 administered IV every 2 weeks in 3 cohorts (cohort 1, 2 and 3 received 0.2, 1.0 and 2.0 mg/kg PRX-102, respectively). Results: Immunogenicity: Three (3) male patients developed treatment induced IgG antibodies to PRX-102 (ADA +); two patients from Cohort 1 and one from Cohort 2. There were no ADA+ patients in Cohort 3. All 3 ADA+ patients became negative in the second year of