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MS assay to detect Tay-Sachs and Sandhoff disease using dried blood spots
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Abstracts/Molecular Genetics and Metabolism 99 (2010) S8–S41
resulting in debilitating pain, organ failure, and premature death. The only effective treatment is enzyme replacement therapy (ERT). In December 2005 the Fabry International Network (FIN) was incorporated as a non-profit organization in the Netherlands. Membership of FIN is open to any patient organization in which Fabry patients are represented (currently 23 in 21 countries) but may be extended by invitation to individuals in exceptional circumstances. FIN has developed a poster to raise awareness of FIN in efforts to educate individual Fabry patient organizations in order to develop a truly worldwide Fabry patient network. The primary aim of FIN is to facilitate collaboration between organizations to support those affected by Fabry disease. It seeks to do this primarily through enabling communication, promoting best practice and acting as an independent forum for Fabry patients around the world. The principle foundation of FIN is to be neutral and independent in all of its communication, action, and decisions. FINs priority continues to be to raise awareness of Fabry disease to ensure all patients worldwide are diagnosed and are provided the best possible comprehensive care for themselves, their families and caregivers. FIN also assists in government advocacy, education and collaborate development of professional and international relationships with all worldwide stakeholders. doi:10.1016/j.ymgme.2009.10.093
Fabry disease (FD) is a progressive, multisystem, X-linked lysosomal storage disorder caused by the deficiency of alpha-galactosidase A. Symptoms and complications of Fabry disease include severe pain in the hands and feet, the inability to sweat, fatigue, a pinkish-purple skin rash, frequent diarrhea, chronic constipation, kidney failure, cardiovascular problems, hearing loss, and depression. Although obstructive pulmonary disease is a recognized symptom of Fabry disease and expert recommendations suggest pulmonary function testing every two years, other pulmonary issues in Fabry patients are not frequently discussed. We describe a 21 year old male with a N215S alpha-galactosidase A mutation who presented with right sided chest pain. Exam revealed decreased breath sounds on the right side and X-ray found a moderate to large right sided pneumothorax. The patient did not exhibit any other signs of respiratory distress. A review of his recent activities found that he had inhaled sharply and blown a shofar (rams horn) during a Yom Kippur service prior to the onset of the chest pain. During surgical repair, it was noted that there were numerous lung blebs. Histopathological examination of lung biopsy found multiple pleural bullae on the pleural surface with unremarkable underlying lung parenchyma. Electron microscopy found ultrastructural features of foamy (lipid-laden) macrophages, but no identifiable zebra bodies or myeloid figures. We suggest that Fabry patients of all ages should be closely monitored for pulmonary complications and spontaneous pneumothorax should be considered in differential for chest pain. doi:10.1016/j.ymgme.2009.10.096
77. The Rare Diseases Clinical Research Networks (RDCRN) Data Management and Coordination Center, J.P. Krischer, University of South Florida, Tampa, FL Jeffrey Krischer, University of South Florida, USA The RDCRN utilizes a Data Management and Coordination Center which provides a secure, customizable, scalable coordinated clinical data management system for the collection, storage, and analysis of diverse data types from clinical researchers working on many different types of rare diseases in geographically disparate locations. The DMCC provides web-based electronic data collection systems, optical scan forms for automated character recognition, interactive voice systems for patient reported outcomes, electronic submission of images (X-rays, CAT scan, MRI, PET, video) and laboratory data from central reference labs worldwide. The USF DMCC web collaboration toolset is a secure portal where RDCRN investigators may post and review documents, announcements, and link to the protocol manager application, training, and other collaboration tools. The tools also provide for a mirrored system to be used for training and demonstrations, access to a web-based media center for live and delayed webcasts of RDCRN educational programming, and a portal for web-based video conferencing. The USF DMCC will maintain data collection and protocol management to identify and track all events specified in RDCRN research protocols. Events include eligibility determination, registration, clinical activities and assessments (e.g., physical exam, medical history), laboratory testing or other investigative procedures, patient selfassessments, and structured patient interviews. Additionally, this system imports laboratory and other data, and manages subject events that are dependent upon test results (e.g., laboratory tests that determine eligibility or establish sub-populations requiring additional follow-up). doi:10.1016/j.ymgme.2009.10.094
78. Environmental and genetic explorations into discordant disease expression in a pair of female monozygotic twins affected by Fabry disease Dawn Laney, Paul Fernhoff, Madhuri Hedge, Chad Alexander, Emory University Fabry disease (FD) is a progressive, multisystem, X-linked lysosomal storage disorder caused by the deficiency of alpha-galactosidase A. Both males and females affected by Fabry disease suffer from significant multisystemic disease symptoms and a reduced quality of life. However, the course of Fabry signs and symptoms are easier to predict in hemizygote males than in heterozygote females. In order to learn more about the expression of Fabry disease in heterozygote females, we describe differences in the phenotypic expression of two 65 year old twins with an I303N mutation through a detailed environmental exposure questionnaire, X-inactivation studies, laboratory studies, biomarkers, clinical evaluation, and genetic testing. doi:10.1016/j.ymgme.2009.10.095
79. Spontaneous pneumothorax in a young man affected by Fabry disease Dawn Laneyb, Paul Fernhoffa, Carlos Abramowskya, Jesse Roman-Rodrigueza, Randolph Hennigara, aEmory University, Atlanta, GA, USA, bChildrens Healthcare of Atlanta, Atlanta, GA, USA
80. A novel mutation in the HEX B gene causing infantile Sandhoff disease: A high throughput semi-automated method for genetic screening of newborns Denis Lehotaya, Braden Fitterera, Nick Antonishyna, Roy Gravelb, Robin Caseyb, Saskatchewan Disease Control Laboratory, Regina, Sask., Canada, bUniversity of Calgary and FootHills Childrens Hospital, Calgary, Alta., Canada a
Sandhoff disease is an autosomal recessive lysosomal storage disorder caused by mutations in the HEXB gene. The HEXB gene conveys the genetic instruction for making the beta subunit of beta-hexosaminidase A and B. The disease leads to ganglioside accumulation in neural cells, neural degradation, and in severe cases early childhood death. The incidence of the disease in northern SK has been abnormally high. Recent genetic sequencing of the HEXB gene from an affected individual has revealed a novel mutation. A retrospective study was performed using dried blood spots (DBS) to assess the allelic frequency of the mutation in the affected population. In the study a real-time PCR assay was developed to detect wild type and mutant alleles present in DBS. A 96-well semi-automated DNA extraction and PCR set-up were utilized to facilitate high throughput rapid screening of a novel mutation for Sandhoffs disease in Saskatchewan for the first time. The heterozygote frequency for infantile Sandhoffs disease in northern Saskatchewan appears to be higher than in the general population. We propose that this method could be incorporated into Saskatchewans neonatal screening program and that it could be adapted for high throughput prospective screening of mutations in communities with high incidence for this disease. doi:10.1016/j.ymgme.2009.10.097
81. A novel, MS/MS assay to detect Tay-Sachs and Sandhoff disease using dried blood spots Denis Lehotaya, Patricia Halla, Michael Gelbb, Denis Lehotaya, aSaskatchewan Disease Control Laboratory, Regina, Sask., Canada, bUniversity of Washington, Seattle, WA, USA An assay was developed to use MS/MS for detecting Tay-Sachs and Sandhoff disease using dried blood spots (DBS). Tay-Sachs and Sandhoff diseases are caused by a deficiency of the enzyme Hexosaminidase (Hex A) and Hex A and B, respectively. Two enzyme substrates were synthesized and used for the assay, one for the measurement of total Hex, and a deuterated version for Hex B. An incubation step to eradicate Hex A allows the measurement of Hex B alone. The deuterated substrate allows determination of both enzymes with one injection. Method development included optimizing buffers, incubation time, and MS/MS conditions. The assay was tested using DBS from patients with Sandhoff disease, as well as unaffected controls. The relative percentage of Hex A increased in adult samples. Samples were analyzed to determine the presence of the disease in northern Saskatchewan, an area where the incidence of Sandhoffs disease is higher than in the general population. The ability of the assay to differentiate heterozygotes from wild type is also being investigated. This new, MS/MS based assay allows for rapid detection of enzyme deficiencies for both Sandhoffs and Tay-Sach disease in DBS, making screening of newborns as part of a newborn screening program possible for the first time. doi:10.1016/j.ymgme.2009.10.098
Abstracts/Molecular Genetics and Metabolism 99 (2010) S8–S41
resulting in debilitating pain, organ failure, and premature death. The only effective treatment is enzyme replacement therapy (ERT). In December 2005 the Fabry International Network (FIN) was incorporated as a non-profit organization in the Netherlands. Membership of FIN is open to any patient organization in which Fabry patients are represented (currently 23 in 21 countries) but may be extended by invitation to individuals in exceptional circumstances. FIN has developed a poster to raise awareness of FIN in efforts to educate individual Fabry patient organizations in order to develop a truly worldwide Fabry patient network. The primary aim of FIN is to facilitate collaboration between organizations to support those affected by Fabry disease. It seeks to do this primarily through enabling communication, promoting best practice and acting as an independent forum for Fabry patients around the world. The principle foundation of FIN is to be neutral and independent in all of its communication, action, and decisions. FINs priority continues to be to raise awareness of Fabry disease to ensure all patients worldwide are diagnosed and are provided the best possible comprehensive care for themselves, their families and caregivers. FIN also assists in government advocacy, education and collaborate development of professional and international relationships with all worldwide stakeholders. doi:10.1016/j.ymgme.2009.10.093
Fabry disease (FD) is a progressive, multisystem, X-linked lysosomal storage disorder caused by the deficiency of alpha-galactosidase A. Symptoms and complications of Fabry disease include severe pain in the hands and feet, the inability to sweat, fatigue, a pinkish-purple skin rash, frequent diarrhea, chronic constipation, kidney failure, cardiovascular problems, hearing loss, and depression. Although obstructive pulmonary disease is a recognized symptom of Fabry disease and expert recommendations suggest pulmonary function testing every two years, other pulmonary issues in Fabry patients are not frequently discussed. We describe a 21 year old male with a N215S alpha-galactosidase A mutation who presented with right sided chest pain. Exam revealed decreased breath sounds on the right side and X-ray found a moderate to large right sided pneumothorax. The patient did not exhibit any other signs of respiratory distress. A review of his recent activities found that he had inhaled sharply and blown a shofar (rams horn) during a Yom Kippur service prior to the onset of the chest pain. During surgical repair, it was noted that there were numerous lung blebs. Histopathological examination of lung biopsy found multiple pleural bullae on the pleural surface with unremarkable underlying lung parenchyma. Electron microscopy found ultrastructural features of foamy (lipid-laden) macrophages, but no identifiable zebra bodies or myeloid figures. We suggest that Fabry patients of all ages should be closely monitored for pulmonary complications and spontaneous pneumothorax should be considered in differential for chest pain. doi:10.1016/j.ymgme.2009.10.096
77. The Rare Diseases Clinical Research Networks (RDCRN) Data Management and Coordination Center, J.P. Krischer, University of South Florida, Tampa, FL Jeffrey Krischer, University of South Florida, USA The RDCRN utilizes a Data Management and Coordination Center which provides a secure, customizable, scalable coordinated clinical data management system for the collection, storage, and analysis of diverse data types from clinical researchers working on many different types of rare diseases in geographically disparate locations. The DMCC provides web-based electronic data collection systems, optical scan forms for automated character recognition, interactive voice systems for patient reported outcomes, electronic submission of images (X-rays, CAT scan, MRI, PET, video) and laboratory data from central reference labs worldwide. The USF DMCC web collaboration toolset is a secure portal where RDCRN investigators may post and review documents, announcements, and link to the protocol manager application, training, and other collaboration tools. The tools also provide for a mirrored system to be used for training and demonstrations, access to a web-based media center for live and delayed webcasts of RDCRN educational programming, and a portal for web-based video conferencing. The USF DMCC will maintain data collection and protocol management to identify and track all events specified in RDCRN research protocols. Events include eligibility determination, registration, clinical activities and assessments (e.g., physical exam, medical history), laboratory testing or other investigative procedures, patient selfassessments, and structured patient interviews. Additionally, this system imports laboratory and other data, and manages subject events that are dependent upon test results (e.g., laboratory tests that determine eligibility or establish sub-populations requiring additional follow-up). doi:10.1016/j.ymgme.2009.10.094
78. Environmental and genetic explorations into discordant disease expression in a pair of female monozygotic twins affected by Fabry disease Dawn Laney, Paul Fernhoff, Madhuri Hedge, Chad Alexander, Emory University Fabry disease (FD) is a progressive, multisystem, X-linked lysosomal storage disorder caused by the deficiency of alpha-galactosidase A. Both males and females affected by Fabry disease suffer from significant multisystemic disease symptoms and a reduced quality of life. However, the course of Fabry signs and symptoms are easier to predict in hemizygote males than in heterozygote females. In order to learn more about the expression of Fabry disease in heterozygote females, we describe differences in the phenotypic expression of two 65 year old twins with an I303N mutation through a detailed environmental exposure questionnaire, X-inactivation studies, laboratory studies, biomarkers, clinical evaluation, and genetic testing. doi:10.1016/j.ymgme.2009.10.095
79. Spontaneous pneumothorax in a young man affected by Fabry disease Dawn Laneyb, Paul Fernhoffa, Carlos Abramowskya, Jesse Roman-Rodrigueza, Randolph Hennigara, aEmory University, Atlanta, GA, USA, bChildrens Healthcare of Atlanta, Atlanta, GA, USA
80. A novel mutation in the HEX B gene causing infantile Sandhoff disease: A high throughput semi-automated method for genetic screening of newborns Denis Lehotaya, Braden Fitterera, Nick Antonishyna, Roy Gravelb, Robin Caseyb, Saskatchewan Disease Control Laboratory, Regina, Sask., Canada, bUniversity of Calgary and FootHills Childrens Hospital, Calgary, Alta., Canada a
Sandhoff disease is an autosomal recessive lysosomal storage disorder caused by mutations in the HEXB gene. The HEXB gene conveys the genetic instruction for making the beta subunit of beta-hexosaminidase A and B. The disease leads to ganglioside accumulation in neural cells, neural degradation, and in severe cases early childhood death. The incidence of the disease in northern SK has been abnormally high. Recent genetic sequencing of the HEXB gene from an affected individual has revealed a novel mutation. A retrospective study was performed using dried blood spots (DBS) to assess the allelic frequency of the mutation in the affected population. In the study a real-time PCR assay was developed to detect wild type and mutant alleles present in DBS. A 96-well semi-automated DNA extraction and PCR set-up were utilized to facilitate high throughput rapid screening of a novel mutation for Sandhoffs disease in Saskatchewan for the first time. The heterozygote frequency for infantile Sandhoffs disease in northern Saskatchewan appears to be higher than in the general population. We propose that this method could be incorporated into Saskatchewans neonatal screening program and that it could be adapted for high throughput prospective screening of mutations in communities with high incidence for this disease. doi:10.1016/j.ymgme.2009.10.097
81. A novel, MS/MS assay to detect Tay-Sachs and Sandhoff disease using dried blood spots Denis Lehotaya, Patricia Halla, Michael Gelbb, Denis Lehotaya, aSaskatchewan Disease Control Laboratory, Regina, Sask., Canada, bUniversity of Washington, Seattle, WA, USA An assay was developed to use MS/MS for detecting Tay-Sachs and Sandhoff disease using dried blood spots (DBS). Tay-Sachs and Sandhoff diseases are caused by a deficiency of the enzyme Hexosaminidase (Hex A) and Hex A and B, respectively. Two enzyme substrates were synthesized and used for the assay, one for the measurement of total Hex, and a deuterated version for Hex B. An incubation step to eradicate Hex A allows the measurement of Hex B alone. The deuterated substrate allows determination of both enzymes with one injection. Method development included optimizing buffers, incubation time, and MS/MS conditions. The assay was tested using DBS from patients with Sandhoff disease, as well as unaffected controls. The relative percentage of Hex A increased in adult samples. Samples were analyzed to determine the presence of the disease in northern Saskatchewan, an area where the incidence of Sandhoffs disease is higher than in the general population. The ability of the assay to differentiate heterozygotes from wild type is also being investigated. This new, MS/MS based assay allows for rapid detection of enzyme deficiencies for both Sandhoffs and Tay-Sach disease in DBS, making screening of newborns as part of a newborn screening program possible for the first time. doi:10.1016/j.ymgme.2009.10.098