this effect. This effect is at least contemporaneous to and compounding to the processof demyelination that characterizes KD. Funded by NIH RNS065808A (ERB).

doi:10.1016/j.ymgme.2009.10.035

19. Carrier testing for MPS I and mucolipidosis II in the IrishTravellingCommunity

Karthryn Bootha,b, Karen Tyleea, Heather Churcha, Fiona Stuarta, Alan Coopera, aWillinkUnit, Genetic Medicine, St. Marys Hospital, Manchester M13 9WL, UK, bClinical Genetics,Belfast City Hospital, Lsiburn Road, Belfast BT 9 7AB, Ireland

The Irish Travelling Community is a distinct population on the island of Ireland.There is a tradition of first cousin marriage within the community. MPS I is presentat a high frequency in this population and Mucolipidosis II is also present withintwo large family within the community.

MPS I was exclusively caused by the common Western European, Caucasianmutation p.W402X. This invariably results in a severe (Hurler) phenotype. Mucolipi-dosis II was due to the common, pan ethnic mutation, c.3503_3504delTC. The WillinkLaboratory in collaboration with Northern Ireland Regional Genetics, has offered pre-natal diagnosis for both conditions to couples from this community. This is performedby assay of lysosomal a-L-iduronidase activity in chorionic villus or cultured amnio-cytes (for MPS I) and assay of multiple lysosomal hydrolases in cultured villus oramniocytes (for Mucolipidosis II). However, such an approach inevitably leads tounnecessary prenatal diagnoses being performed.

Once the underlying genetic lesions had been identified, carrier testing for bothconditions has been offered, either prior to conception or early in pregnancy. Bothmutations were identified by sequencing.

One hundred and ninety-seven relatives have been screened for p.W402X and 60for c.3503_3504delTC. 101 carriers of p.W402X and 25 carriers of c.3503_3504delTChave been identified. In this limited sample, two individuals were identified who car-ried both the p.W402X and c.3503_3504delTC mutations. This has allowed prenataldiagnosis to be offered only when a pregnancy is truly at risk for one of these twoconditions.

doi:10.1016/j.ymgme.2009.10.036

20. Cardiac valvular interstitial cells in MPS I

Elizabeth Braunlina, Jakub Tolara, Shannon Mackey-Bojackb, Tiwanda Marshc, PaulOrcharda, Frederick Schoend, aUniversity of Minnesota, Minneapolis, MN, USA, bThe JesseE. Edwards Registry, St. Paul, MN, USA, cNational Institute of Environmental HealthScience, Research Triangle Park, NC, USA, dHarvard Medical School, Boston, MA, USA

Background: Cardiac valve thickening by glycosaminoglycan-laden (GAG-laden)cells is clinically significant both in native mucopolysaccharidosis type I (MPS I)and despite hematopoietic stem cell transplantation or enzyme replacement therapy.

Hypothesis: We hypothesized that the GAG-laden cells in MPS I cardiac valves areactivated valvular interstitial cells (VICs), similar to those in valvular disease, adapta-tion to altered loading and living tissue valve replacements.

Methods: We performed pathologic analysis on cardiac atrioventricular valve tis-sue (two mitral, one tricuspid valve) from two infants with untreated MPS I and com-pared the results to mitral valve tissue (n = 2) from age-matched normal controls.

Results: We identified abundant small, spindle-shaped cells characteristic of rest-ing VICs within all layers of normal valve tissue. In MPS I, the deep cells within thesame layers of valve tissue were uniformly swollen with clear cytoplasm and eccen-tric nuclei. Immunohistochemical studies showed negative staining by these cells forCD68, CD3, and CD31 (indicating that they were not histiocytes, lymphocytes or vas-cular endothelial cells) and positive staining for vimentin, alpha-smooth muscle actin,and matrix-metalloproteinase-9, characteristics of activated VICs.

Conclusions: GAG-laden VICs within the cardiac valves of MPS I individuals have aphenotype similar to that of activated VICs, described in other models of extracellularmatrix remodeling and response to injury. This suggests that these cells may be con-tributing to valve dysfunction beyond accumulating GAG, and that such mechanismsmay be effective targets for inhibiting the deleterious effects of MPS I-related valvepathology.

doi:10.1016/j.ymgme.2009.10.037

21. Glycan-based biomarkers for the mucopolysaccharidoses

Jill Browna, J.R. Browna, R. Carrolla, N. Tambea, C. Glassa, K. Langfordb, S. Lec, A.Victoroffc, J.E. Wraithb, P. Dicksonc, B.W. Biggerb, B.E. Crawforda, aZacharon

Pharmaceuticals, San Diego, CA, USA, bUniversity of Manchester, Manchester, UK,cHarbor UCLA Medical Center, Torrance, CA, USA

The mucopolysaccharidoses (MPS) are caused by a deficiency in one of theenzymes responsible for the degradation of glycosaminoglycans (GAG). We havedeveloped biomarker detection methods based on the accumulation of glycan sub-strates. Many biomarkers have been considered including enzyme activity, proteinand mRNA markers, and measures of substrate accumulation. Quantification of sub-strate accumulation is potentially the most powerful approach given its direct rele-vance to pathology. However, historical methods to measure substrateaccumulation in lysosomal storage disease (LSD) have been hampered by the pres-ence of endogenous and extremely heterogeneous substrate material. To overcomethis challenge, the Sensi-Pro Substrate Assay was developed. By selectively amplifyingthe substrates accumulating due to the deficient enzyme, this method provides anassay with unique capabilities that can be applied across many (LSD). To validatethe method, we analyzed substrate in tissue and fluid samples from MPS subjects.Samples of serum, plasma, urine, and cerebrospinal fluid were obtained from MPSpatients, MPS animals, and normal subjects. The results demonstrate that the assaycan sensitively detect GAG accumulation with substantial variation in GAG levels insamples from MPS subjects compared to normal subjects. Response to enzymereplacement therapy was also demonstrated, and sample sizes of less than 50 l wererequired. At time of abstract submission, additional data collection to correlate theGAG levels to clinical endpoints are in process, creating the opportunity for a vali-dated biomarker of MPS disease severity and response to therapy in a variety of sam-ple types.

doi:10.1016/j.ymgme.2009.10.038

22. Small molecule inhibitors of glycosaminoglycan biosynthesisas substrateoptimization therapy for the mucopolysaccharidoses

Jill Brown, J.R. Brown, R. Carroll, C. Glass, B.E. Crawford, Zacharon Pharmaceuticals,San Diego, CA, USA

The mucopolysaccharidoses (MPS) are a family of lysosomal storage diseasescaused by a deficiency in enzymes responsible for the degradation of glycosaminogly-cans (GAG). Substrate optimization therapy (SOT) is a novel therapeutic approach forlysosomal storage disease based on selectively modifying the substrates to renderthem more readily degraded despite the presence of specific enzyme deficiencies,without reducing the overall amount produced or altering normal glycan function.In MPS, this is accomplished with small molecules targeting specific GAG biosyntheticenzymes, thus selectively and favorably modifying the glycan sulfation pattern. Toidentify drug candidates, we screened a library of 74,000 drug-like small moleculesfor modifiers of heparan sulfate biosynthesis using cell-based high throughput assaysand glycan structural analysis technology. Of the 264 hit compounds identified in theprimary screens, 30 were found to modify heparan sulfate biosynthesis in culturedcells. Four of these compounds reduce GAG accumulation in primary human fibro-blasts from MPS patients, and glycan structural analysis revealed changes consistentwith the intended SOT mechanism. At time of abstract submission, ongoing studiesare focused on analog design, synthesis, and testing to improve compound potency,and subsequent studies are planned to achieve the following preclinical product char-acteristics: (1) alteration of glycan biosynthesis to allow substrate degradationdespite presence of enzyme deficiencies in MPS, (2) efficacy in MPS animal models(potentially several MPS classes), (3) CNS penetrant, (4) pharmacokinetic profile sup-portive of convenient dosing, and (5) favorable safety profile.

doi:10.1016/j.ymgme.2009.10.039

23. Fabry disease identification

Marsha Browning, MGH/Harvard Medical School, Boston, MA, USA

Fabry disease is a treatable but clinically under recognized X-linked lysosomalstorage disease. High-risk populations, including patients with ischemic stroke andcardiovascular defects, if screened appropriately in a high-throughput, cost-effectivemanner, would demonstrate a higher prevalence of Fabry disease than previouslyreported in restricted patient populations. The prevalence of undiagnosed Fabry inend-stage renal disease, including dialysis and kidney transplant recipients, is 20–50 higher than that of the general population. An aggressive detection platform isneeded to identify previously undiagnosed or misdiagnosed patients, including arobust means to detect female Fabry patients, a previously underserved componentof these at-risk groups. The specific aims of this project are to: (1) To develop ahigh-throughput, cost-effective combined biochemical and molecular testing plat-form for Fabry Disease from blood and urine filter paper collection that can be adapt-

S12 Abstracts/Molecular Genetics and Metabolism 99 (2010) S8–S41

able nationally and internationally for a broader epidemiologic study of detection effi-ciency. (2) To pilot the most expansive program for Fabry Disease detection to date inat-risk populations (stroke and cardiac patients from Texas and Massachusetts; end-stage renal disease patients from Minnesota) with the capability of including femalepatients. (3) To improve diagnostic ascertainment, education, and awareness of FabryDisease. Dr. Brownings group has developed a modified enzyme analysis using a fluo-rescence based high-throughput microplate (96 well) method as a fully automatedsystem. If scanning is abnormal, full sequence analysis will be performed. Lowenzyme levels automatically reflex to full gene sequencing (estimated to be <20% ofall samples when tiered through enzyme/scanning processes). Confirmatory molecu-lar testing entails full sequencing of the GLA gene using ABI PRISM 3710 sequencer(Applied Biosystems). A database assembly of mutations and polymorphisms willbe maintained. Dr. Schiffmanns group has recently developed and validated a methodfor measuring Gb3 using ultra-performance liquid chromatography (UPLQ–MS/MS inwhole urine-soaked filter paper. The method uses multiple reaction monitoring posi-tive ion electrospray ionization with a total run of 3 min and results expressed in u,gGb3/mmole of creatinine. Three at-risk groups will be screened. (1) End-stage renaldisease patients in Minnesota from a pool of 7347 patients. From the study question-naire and the patients clinical and demographic features, we will develop predictivemodels allowing >50% reduction in subjects to be screened with a <5% loss of identi-fied Fabry patients in order to develop an efficient national end-stage renal diseaseand family screening strategy. (2) 3700 ischemic stroke patients have been identifiedthrough the Harvard Partners RDPR system, and between 3000 and 4000 strokepatients have been identified through the Texas Heart program. All patients withischemic stroke above 1-year-of-age in the stroke clinics through both institution willbe offered clinical testing for Fabry disease as a part of routine care. Positive predic-tive values and prevalence rates will be established linking the biomarkers andgenetic mutations via a joint database analysis. (3) 5800 cardiac patients have beenidentified through the Harvard Partners RDPR system, and between 8000 and10,000 stroke patients have been identified through the Texas Heart program for‘‘at-risk’’ Fabry conditions, including hypertrophic cardiomyopathy, arrhythmias,unexplained valvular disease, or unexplained coronary artery disease. All cardiacpatients at both institutions will be offered clinical testing for Fabry disease as a partof routine care. Positive predictive values and prevalence rates will be establishedlinking the biomarkers and genetic mutations via a joint database analysis.

doi:10.1016/j.ymgme.2009.10.040

24. The prevalence and characterization of respiratory involvement in patientswith Hunter syndrome in the Hunter Outcome Survey

Barbara Burtona, Laurie Smithb, Roberto Giuglianic, Erlane Ribeirod, Julian Raimane,aNorthwestern Universitys Feinberg School of Medicine, Chicago, IL, USA, bChildrens MercyHospitals and Clinics, Kansas City, MO, USA, cHospital de Clnicas de Porto Alegre, PortoAlegre, Brazil, dFaculdade de Medicina de Juazeiro do Norte, Juazeiro do Norte, Brazil,eUniversity of Toronto, Toronto, Canada

Hunter syndrome is an X-linked disease of glycosaminoglycan (GAG) catabolismcaused by a deficiency in the lysosomal enzyme, iduronate-2-sulfatase. ProgressiveGAG accumulation within tissues and organs contributes to the multi-system pathol-ogies characteristic of the disease. The Hunter Outcome Survey (HOS) is a global data-base that is used to characterize the natural history of the disease as well as toevaluate the safety and effectiveness of enzyme replacement therapy (ERT) with idur-sulfase (Elaprase, Shire HGT, Cambridge, MA, USA). As of July 2009, 579 prospectivepatients were enrolled in HOS (median age at last examination was 10.7 years;10th–90th percentile = 3.9–23.9 years). Of the 522 patients with available data, 85%had at least one respiratory finding from birth to last visit in HOS. Sleep apnea wasreported for 39% of patients with a median age of onset of 6.6 years, and the use ofCPAP, BiPAP, or mechanical ventilation was reported for 11% of patients with a med-ian age at first intervention of 13.9 years. Other commonly reported findings includedobstructive airway disease (25%) and dyspnea (23%). Average percent predictedforced vital capacity (%FVC) was about 65% in 74 patients for whom pre-ERT testresults were available and was not significantly associated with age. In contrast,the percent FVC expired in the first second significantly decreased with age, suggest-ing progression of restrictive/obstructive airway disease. These respiratory findingsconfirm their major contribution to the morbidity of Hunter syndrome.

doi:10.1016/j.ymgme.2009.10.041

25. Longitudinal studies of the glycoproteinoses: An international update

Sara Catheya,e, Callum Wilsonb, David Sillencec, Lucia Horowitza, Jenny Nobled,aGreenwood Genetic Center, bStarship Hospital, Auckland, New Zealand, cUniversity ofSydney, Childrens Hospital at Westmead, Australia, dLysosomal Diseases, New Zealand,eISMRD – The International Advocate for Glycoprotein Storage Diseases

The diseases characterized by impaired degradation of glycoproteins within thelysosomes include a-mannosidosis, aspartylglucosaminuria, b-mannosidosis, fucosi-dosis, galactosialidosis, mucolipidosis II, mucolipidosis III, Schindler disease, and sial-idosis. Even collectively these diseases are rare, prompting a global approach tolongitudinal studies of these conditions.

Patient advocacy groups and family support networks around the world havechampioned natural history studies of each condition. Lysosomal Diseases New Zea-land (LDNZ), ISMRD – The International Advocate for Glycoprotein Storage Diseases,the Australian MPS Society, and the (US) National MPS Society have supported partic-ipation in these studies among their member families. In New Zealand and Australiaall families known to be affected by one of the glycoproteinoses have been invited toparticipate in special clinics to be held November 2009 in Sydney, Australia and Auck-land, New Zealand. Individuals with mucolipidosis II and III a/b, aspartylglucosamin-uria, a-mannosidosis, sialidosis, and fucosidosis, will participate.

The natural history protocols focus on prenatal and postnatal growth, neurode-velopmental progress, physical findings (craniofacial and skeletal dysmorphology,visceral involvement, neurological signs), cognitive skills, medical complications,and survival. Detailed histories, physical examinations, and psychoeducational evalu-ations are performed.

Insights gained from the patients in New Zealand and Australia will be includedin this progress report of the longitudinal studies of the glycoproteinoses.

doi:10.1016/j.ymgme.2009.10.042

26. A re-analysis of disease stage progression in Krabbe disease (infantile globoidcell leukodystrophy, iGLD)

Lawrence Charnas, Nitin Nair, Robert Mensah, Shire HGT, Cambridge, MA, USA

Background: iGLD is a rapidly progressive autosomal recessive neurodegenerativedisorder caused by deficiency of the lysosomal acid hydrolase galactosylceramidase.Although the clinical classification into three disease stages has broad acceptance[Hagberg etal., 1969. Neuropdiatrie 1, 74–88], the time of progression between dis-ease stages has not been quantified and the correlation of individual components ofthe composite endpoints with their assigned disease stage have not been replicated.Precise clinical definitions of each stage and accurate progression rates are critical tothe success for a therapeutic development program.

Objectives: To estimate the time of transition between disease stages using pub-lished data.

Methods: Analog data published by Hagberg (op. cit.) were manually digitized anda survival curve created using the Kaplan–Meier method.

Results: The duration of each stage is approximately 3 months. The mean durationof Stage 1 was 3.4 1.9 months, median 3 months (range 1–8). For Stage 2, the meanduration was 3.2 2.1 months, median 2.5 months (range 1–7.5), and for Stage 3, themean duration was 4.2 3.7 months, median 2.75 months (range 0.5–14). The mean(and median) calculated survival time after disease onset was 11 months (SEM = 1)identical to survival time reported in Hagbergs paper when adjusted for censoring.Both the mean and median survival of Hagbergs cohort were substantially shorterthan that from a more recent registry [Duffner etal., 2009. Ped Neuro 40, 13–18],mean survival 21 months (SEM = 3), median 17 months.

Conclusions: iGLD is rapidly progressive, with a median time from symptom onsetto Stage 3 of about six months. Survival of affected infants has increased substantiallybetween 1969 and 2009 despite the lack of specific therapy. This may reflect changesin supportive medical care. Development of specific therapeutic products for iGLD inthe current medical era require validation of the clinical components comprisingeachdisease stage as well as the rate of changes that accurately reflect diseaseprogression.

doi:10.1016/j.ymgme.2009.10.043

27. A study of intrathecal enzyme replacement for cognitivedecline in muco-polysaccharidosis I

Agnes Chena, Patricia Dicksona, Elsa Shapirob, Barbara Lyonsa, Shih-hsin Kana, DanielGuillaumec, aLos Angeles Biomedical Institute at Harbor – UCLA Medical Center, Torrance,CA, USA, bUniversity of Minnesota, Minneapolis, MN, USA, cOregon Health SciencesUniversity, Portland, OR, USA

Patients with mild and moderate forms of MPS I have been found to suffer fromdeclines in memory and intelligence and have essentially no treatment for theseproblems. Enzyme replacement therapy has been shown to reduce lysosomal storage,and when given intrathecally, has been shown to achieve high levels of iduronidaseand reduce GAG storage to normal in the brain, spinal cord and spinal meninges inthe canine model. Several MPS I patients have been treated with intrathecal rhIDUin an ongoing trial for spinal cord compression. This study will further investigate

Abstracts/Molecular Genetics and Metabolism 99 (2010) S8–S41 S13