The LEIM performs the diagnosis of PD in DBS according to thetechnique described by Chamoles et al. (2004), which results areexpressed both in a Neutral Glicosidase/Acid Glicosidase ratio (N/A)and by the percentage of inhibition of Neutral Glicosidase (% INH)using acarbose. Values of N/A>40 and %INH>87 establish thediagnosis of PD. We retrospectively analyzed the informationprovided on the requisition form for each sample referred to us forinvestigation of PD from 2006 to 2010. From December/2006 toDecember/2010 we had received 734 samples for investigation of PDin DBS. Of all samples, 6 had low quality and we requested a newsample. After the analysis, 39 (5.5%) samples were in the affectedrange, 12 (1.7%) were in a borderline range (N/A>40 and %INH<87or N/A<40 and %INH>87), and 654 confirmed negative. The N/A(mean+−SD) and the % INH (mean+−SD) of the positive,borderline and negative samples were respectively 67.94+−17.97and 91.54+−2.49, 38.43+−9.84 and 87.88+−3.41, and 13.1+−5.1and 67.14+−11.24. The N/A could clearly distinguish the 3 groupswithout overlapping with statistical significance. Among the borderlinegroup, samples with N/A=30-40 were from individuals with moremuscular symptoms. Our results showed that in most of cases the DBSmethod alone established the definite diagnosis of PD, and in 12 casesthe result was within the borderline range. These results could beexplained by the individual biochemical variation, pointingout the needof other techniques in such cases. Financial support: FAPESP, CNPq,CAPES, AFIP and IGEIM.

doi:10.1016/j.ymgme.2011.11.101

Dyslipidemia and Fabry Disease

Dawn Laney a, Paul Fernhoff a, Chardae' Hill b, Mary Ellen Yupari c,Suma Shankar a, Muhammad Ali Pervaiz a, Robin Thompson a, aEmoryHealthcare, Decatur, GA, USA, bClayton State University, Clayton, GA,USA, cGeorgia State University, Atlanta, GA, USA

Objective: To evaluate the fasting lipid profiles in a cohort of Fabrydisease (FD) patients.

Background: Fabry disease (FD) results from an inability toeffectively metabolize globotriaosylceramide (GL3) and is associatedwith premature vascular disease. There is no known associationbetween FD related vascular disease and concomitant dyslipidemia;however, dyslipidemia is a known modifiable risk factor thataccelerates cardiovascular disease.

Methods: We reviewed the fasting total cholesterol (TC), high-density lipoprotein (HDL), and lower-density lipoprotein (LDL) of 78Fabry patients. Of these we excluded any individuals on cholesterollowering medications (n=9) or under the age of 20 years of age(n=11). The remaining patient population under study includes 58adult subjects (mean age 40.8; age range: 20–78).

Results: Adult patients had mean TC levels of 176.3 mg/dl, meanHDL levels of 56.2 mg/dl, and mean LDL 97.1 mg/dl. Only one 47 yearold male patient had a TC level less than 100 mg/dl. 19/52 adultpatients had optimal HDL values and 29/52 adult patients hadoptimal LDL levels.

Discussion: The majority of adult Fabry patients under study hadnormal lipid profiles. This suggests that Fabry disease does notpredispose this patient population to dyslipidemia as a compoundingrisk factor for cardiovascular disease. However, there does seem to bean identifiable subset of patients with a consistently optimal LDLlevels that provides a negative risk factor of cardiovascular disease.

doi:10.1016/j.ymgme.2011.11.102

Tandem Mass Spectrometry Multiplex Analysis of Lyso-Gb3-Related Analogs for Fabry Disease

Pamela Lavoie, René Gagnon, Michel Boutin, Félix Dupont, ChristianeAuray-Blais, University of Sherbrooke, Faculty of Medicine and HealthSciences, Sherbrooke, Quebec, Canada

Introduction: Fabry disease is a multisystemic, X-linked lysosomalstorage disorder leading to accumulation of glycosphingolipids inbiological fluids. Two specific Fabry disease-related biomarkers havebeen evaluated and quantified in plasma and urine of Fabry patients:globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). The objectives were to devise and validate a liquid chromato-graphy-tandem mass spectrometry (LC-MS/MS) method for thequantification of 7 urinary lyso-Gb3-related analogs for Fabry diseaseto eventually evaluate correlations with disease progression andseverity.

Method: Urine samples of Fabry patients and healthy age-matchedcontrols were acidified, extracted with Oasis-MCX cartridges (WatersCorp.) and evaporated under nitrogen. Samples were reconstituted in50% acetonitrile/water/0.1% formic acid, and injected in the HPLCAlliance 2795XE coupled to a Quattro Micro tandem mass spectro-meter (Waters). Chromatographic separationwas performed using anAtlantis HILIC Silica column (Waters). Lyso-Gb3 analogs wereanalyzed in positive electrospray, MRM mode using an internalstandard (IS). Results were processed with MassLynx (Waters).

Results: Seven urinary lyso-Gb3 analogs at mass/charge (m/z) of:m/z 758; m/z 774; m/z 784; m/z 800; m/z 802; m/z 820; and m/z 836were quantified. We developed a chromatographic run with a similarretention time for all analogs with only one IS. Urine samples werenormalized to creatinine. Lyso-Gb3 analogs were not detected incontrol samples. Preliminary results show that male Fabry patientspresent higher lyso-Gb3 analogs excretion levels compared to femalesand these levels are reduced after enzyme replacement therapytreatment.

Conclusions: We devised a simple and efficient multiplex methodto quantify 7 lyso-Gb3 analogs by LC-MS/MS.

doi:10.1016/j.ymgme.2011.11.103

SBC-103, aRecombinantEnzymeReplacementTherapy,DemonstratesPotential for the Treatment Of Sanfilippo Syndrome Type B

Mark Leavitt, Wei Hu, Robert Lyng, Rachel Richard, Zhinan Xia, SaraMathews, Marla Popov, Anthony Quinn, Synageva BioPharma, Lexington,MA, USA

Mucopolysaccharidosis IIIB (Sanfilippo syndrome type B) is alysosomal storage disorder caused by deficiency in the enzymealpha-N-acetyl-glucosaminidase (NAGLU). At present there is notreatment for this disease. Recombinant lysosomal enzymes aredifficult to produce and significant manufacturing challenges haveresulted in supply limitations for some existing treatments. Earlyattempts to produce rhNAGLU in cell culture for use as an enzymereplacement therapy (ERT) failed due to an inability to produceenzyme with properties that allowed uptake into key target cells(Weber B et al., 2001). We have previously described methodologiesfor the consistent production of SBC-102 (a recombinant humanlysosomal acid lipase which is now in clinical trials) with efficient invitro uptake and a high level of efficacy in a preclinical diseasemodel.

The human NAGLU coding sequence was cloned into a retroviralvector designed to express the gene in oviduct cells of hens resultingin high levels of expression of NAGLU in egg white (EW). rhNAGLU

Abstracts / Molecular Genetics and Metabolism 105 (2012) S15–S69 S43

activity was confirmed in EW, and subsequent experiments demon-strated the successful integration of rhNAGLU gene and the promoterelements into a single integration site. rhNAGLU purified from EWusing column chromatographic methods demonstrated good uptakeinto fibroblasts from a patient with MPS IIIB deficiency withcorrection of enzyme deficiency.

Conclusions: SBC-103, a rhNAGLU expressed in EW has propertieswhich allow effective lysosomal targeting to key target cells incontrast to enzyme produced using cell culture. These studiesindicate that SBC-103 warrants further investigation as a therapyfor patients with Sanfilippo syndrome type B.

doi:10.1016/j.ymgme.2011.11.104

BMN 110 (Recombinant Human GALNS), an InvestigationalEnzyme Replacement Therapy for Mucopolysaccharidosis TypeIVA (MPS IVA Or Morquio Syndrome), Promotes Type II CollagenSynthesis in MPS IVA Patients

Florence Lorget a, Sungwook Lee a, Kelly Lau a, Laurie Tsuruda a, ChrisHendriksz b, Simon Jones c, Ashok Vellodi d, Sabrina cheng a, CelesteDecker a, Charles O'Neill a, aBioMarin Pharmaceuticals Inc., Novato, CA,USA, bBirmingham Children's Hospita, Birmingham, UK, cCentralManchester University Hospitals, Manchester, UK, dGreat Ormond StreetHospital, London, UK

Mucopolysaccharidosis type IVA (MPS IVA) or Morquio syndrometype A, is a lysosomal storage disorder caused by a deficiency of theenzyme N-acetylgalactosamine-6 sulfatase (GALNS) that results inaccumulation of glycosaminoglycan keratan sulfate (KS) in hyalinecartilage, tissue macrophages, heart valves and cornea. KS accumula-tion in the hyaline cartilage leads to a profound skeletal dysplasiaattributed to impaired chondrocyte proliferation and differentiation.During the Dose Escalation Phase 1/2 clinical trial, 20 patients, aged 5to 16 years old, were administered weekly BMN 110 infusions at the0.1 mg/kg dose level (Weeks 1 to 12), 1 mg/kg dose level (Weeks 13to 24), 2 mg/kg dose level (Weeks 25 to 36) and 1 mg/kg dose level(Weeks 37 to 72) during the Continuation Period. Type IIA Collagen Npropeptide (PIIANP), a marker of collagen type II formation, wasassessed in the serum of the patients prior to dosing at baseline andWeeks 13, 25, 37 and 72. A marked increase in mean PIIANP levels incomparison to baseline levels was measured at Week 25 (+39.8%,p=0.058), Week 37 (+65.6%, p<0.001) and Week 72 (+84.5%,p<0.001). As resting and proliferating chondrocytes are the mainsource of collagen type II, this data suggests that BMN 110 afterintravenous infusions distributes to the cartilage and promotes thechondrocyte function, thus potentially improving the systemicskeletal dysplasia in MPS IVA patients.

doi:10.1016/j.ymgme.2011.11.105

Evaluation of Plasma Globotriaosylsphingosine in Patients withAnderson-Fabry Disease in Brazil on Enzyme Replacement Therapywith Agalsidase Alfa

Charles Lourenco a, Janice Coelho b, Miguel Moyses Neto a, OsvaldoMerege Vieira Neto a, Wilson MarquesJr. a, c, aUniversity of Sao Paulo,Ribeirao Preto, Sao Paulo, Brazil, bFederal University of Rio Grande do Sul,Porto Alegre, Brazil, cUniversity of Sao Paulo, Sao Paulo, Brazil

Introduction: Anderson-Fabry disease (AFD) is an inheriteddisorder due to an enzymatic deficiency of alpha-galactosidaseactivity, resulting in glycosphingolipid accumulation. As enzyme

replacement therapy (ERT) involving recombinant enzyme has beenintroduced for this disease, finding a reliable surrogate biomarker formonitoring the therapy is very important for evaluation of benefitsand better follow-up of patients on ERT.

Material and Methods: We studied 6 Fabry disease patients (fourmales and two females) over a period of 6 months regarding diseaseprogression and clinical outcome under ERT. Plasma globotriaosyl-sphingosine (lysoGb3) levels were measured in dried blood spotsbefore and 6 months after treatment with agalsidase alfa.

Results: Both male and female patients had elevated levels oflysoGb3; in the males subjects, the increase was impressive beforestarting treatment. After 6 months, plasma lyso Gb3 were reduced inall patients; in males, plasma lyso Gb3 dropped more dramaticallythan in the females subjects. Pre-treatment plasma lysoGb3 con-centrations of Fabry females were relatively low but remained stableor decrease after ERT. At baseline, only only two patients showedabnormal cardiac findings (mild LVH and mitral valvulopathy,respectively). Brain MRI with angiography was abnormal in twopatients (multiple hypersignal loci with arterial tortuosity andperiventricular white matter changes). Proteinuria and microalbu-minuria were present in all patients; after 6 months under ERT, brainMRI changes remained stable and no further kidney deteriorationwasseen. Cardiac evaluation still showed no abnormal findings in theother patients and remained unchanged in the other two.

Conclusion: Our cohort of Anderson-Fabry disease patientsshowed reduction of plasma lysoGb3 after ERT introduction.Although it was not possible to evaluate differences in other systemsbecause of the short period of observation, our finding confirmsprevious studies which show that ERT can correct plasma lysoGb3 inFabry disease patients even in a short period of 6 months.

doi:10.1016/j.ymgme.2011.11.106

Niemann-Pick Type C in Brazil: Natural History and Clinical Coursein 42 Patients

Charles Lourenco a, Fernanda Timm b, Roberto Giugliani b, WilsonMarquesJr. a, aUniversity of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil,bFederal University of Rio Grande do Sul, Porto Alegre, Brazil

Background: Niemann-Pick type C disease (NPC) is a rentlessneurodenerative disorder characterized by a defect in cholesteroltrafficking; it is a complex disorder with wide range of clinicalphenotypes. Here we describe clinical characteristics and naturalhistory of 42 Brazilian NPC patients.

Methods:Retrospective studyandreviewof laboratory/neuroimagingdata were carried out in NPC patients diagnosed in the last 6 years.

Results: Twenty-six patients were confirmed to have NPC by filipinstaining (14 patients required molecular analysis). Regarding clinicalform, 7 were perinatal, 16 infantile, 11 juvenile and 8 adults.Prolonged neonatal jaundice was a common feature and five of themwere diagnosed with “neonatal hepatitis”. Hepatosplenomegaly waspresent in all perinatal/infantile patients, but absent in 6 adults.Ocular abnormalities were seen in 38 patients (mostly, verticalsupranuclear gaze paralysis). Leukoencephalopathy and progressivecerebral/cerebellar atrophy were the main MRI features. Othersystemic manifestations included dystonia, cataplexia, immunodefi-ciency, dysphagia. At the time of the study, 13 patients were deceased.

Conclusions: Onset of neurological disease in NPC patients isextremely variable, even in the same sibship. Better understanding ofthe natural history of the disease is crucial for evaluation of potentialtherapeutic approaches in such devastating disorder.

doi:10.1016/j.ymgme.2011.11.107

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