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  1. 1. PLATFORM PRESENTATIONS BIOCHEMICAL GENETICS 25 26 Fabry disease presenting in the pediatric age group:clinical and ethical con- The Phase 4 Clinical Trial Demonstrates Clinical Benefit of Fabrazyme® (agal- cerns. L.A. Clarke1,2, J. Barranger2, R. Hopkin2, M. Banikazemi2, J. Charrow2, sidase beta). R.J. Desnick, Phase 4 Investigators of the International Fabry Disease C.M. Eng2, G. Pastores2, C.R. Scott2, K. Sims2, D. Warnock2, W. Wilcox2. Study Group. Dept Human Genetics, Box 1498, Mount Sinai School of Medicine, 1Department of Medical Genetics, University of British Columbia, Vancouver, Brit- New York, NY. ish Columbia, Canada; 2North American Board of Medical Advisors, Fabry Fabry disease, an X-linked recessive lysosomal storage disorder, is caused by de- Registry. ficient activity of α-galactosidase A and the resultant accumulation of globotriaosyl- The advent of enzyme replacement regimes for Fabry disease and the realization ceramide (GL-3) and related glycosphingolipids particularly in endothelial cells of that therapy instigated early in the course of disease may lead to improved outcomes the heart and kidney. Enzyme replacement therapy (ERT) with Fabrazyme® for has sparked interest in the pediatric presentation of Fabry disease. As of Nov. 2004 Fabry disease was approved in Europe (2001) and the United States (2003) after a the Fabry disease registry contained data on 1065 individuals with Fabry disease, Phase 1/2 clinical trial confirmed the dose-dependent effectiveness of ERT (Eng et 110 (10.3 %)were in the pediatric age group(<18 years). There were 70 males al., Am J Hum Genet 68:711-722, 2001) and a Phase 3 clinical trial (Eng et al., N (64%)and 40 females(36%). The median age of onset of first symptoms was 5.5 yrs Engl J Med 345:9-16, 2001) and extension study (Wilcox et al., Am J Hum Genet for the pediatric males (n=48)and 8.0 yrs for the pediatric females (n=13). 55(79%) 75:65-74, 2004) showed that ERT safely reversed the pathogenic GL-3 accumula- of the males <18 yrs indicated having Fabry symptoms as opposed to 17 (43%)of the tion in heart, kidney, and skin during the three year study period. A Phase 4 trial was females < 18 yrs. For those patients <18 yrs with symptoms,the most common pre- required by the accelerated approval program of the FDA to further verify and con- senting symptom in males was pain(44/55, 80%)followed by gastrointestinal (18/55, firm clinical benefit. This study was a multinational, multicenter, double-blind, pla- 33%)and angiokeratoma (12/55, 22%). Interestingly,symptomatic females had a cebo-controlled trial involving 82 patients with Fabry disease who had mild to similar profile: pain(14/17,82%),gastrointestinal(6/17, 35%)and angiokeratoma (3/ moderate renal disease. The patients were randomized 2:1 (Fabrazyme®: placebo) 17, 18%). No patient <18 yrs presented with cardiac or cerebrovascular symptoma- at each study site and the median study time was 18.5 months (35 months total). The tology. 437 adult (≥18 yrs of age) registry patients reported presenting symptoms in primary endpoint compared the time to the first clinical event (renal, cardiac, cere- the pediatric age range,301 male (69%)and 136(31%)female. Males reported pain as brovascular or death) between the two treatment groups. After a clinical event, a pa- a presenting feature,81% (243/301)followed by angiokeratoma, 36%(108/301) and tient could be switched to open-label Fabrazyme®. The Phase 4 clinical trial showed gastrointestinal, 24%(73/301). Females also reported similar findings with pain that compared to placebo, Fabrazyme® at 1 mg/kg every two weeks slowed the rate 81%(110/136), angiokeratomas 16% (22/136), and gastrointestinal 15%(21/136)the of progression of Fabry disease and substantially reduced the risk of all events (renal, most common presenting features. Interestingly,the median age of first symptoms cardiac, cerebrovascular) as well as the renal, cardiac or cerebrovascular events in- for these adult males was 8.0 yrs (n=301) and 10.0 yrs (n=136) for females. dividually. Of the 82 patients in the study (the Intent to Treat population), the pa- Conclusion:It is likely that Fabry disease is under diagnosed in the pediatric popu- tients who received Fabrazyme® were 43% less likely than the placebo-treated lation, and the symptom severity of young patients has been under estimated. The patients to experience a clinically significant renal, cardiovascular or cerebrovascu- data obtained from adults and children indicates that pain, angiokeratoma and gas- lar event. After adjusting for a baseline imbalance in proteinuria between the two trointestinal symptomatology are common in pediatric Fabry disease. Registry data treatment groups, patients randomized to Fabrazyme® were 53% less likely to ex- indicates that females are commonly affected during the pediatric age and have sim- perience a clinically significant event. Similar to other renal diseases, baseline pro- ilar presenting signs to the males, despite their milder renal disease in adulthood. The teinuria was the most important determinant of outcome. Among the 74 patients who data indicates that pediatric patients with Fabry disease require detailed assessment were compliant with the study protocol (the Per-Protocol population), after adjust- particularly with respect to pain, gastroenterological symptoms and dermatological ment for a baseline imbalance in proteinuria between the two treatment groups, the findings. In addition, pediatric neurology, dermatology and gastroenterology clinics patients who received Fabrazyme® were 61% less likely to experience a clinically should be familiar with the presenting signs of Fabry disease. Most importantly, significant event (p=0.034). The most pronounced benefit of Fabrazyme® was seen pediatric Fabry patients should be specifically counseled with respect to the present- when therapy was started earlier in the course of the disease (i.e., with less renal dys- ing signs of disease so as to avoid anxiety and unnecessary investigations related to function). These findings emphasize the importance of early treatment with 1 mg/kg early signs and symptoms. of Fabrazyme®. BIOCHEMICAL GENETICS 27 28 Disease Management and Therapeutic Goals for Gaucher Disease. G.A. Identification of Protein Biomarkers for Achlorhydria in Mucolipidosis IV. Grabowski1, G.M. Pastores2, N.J. Weinreb3, H. Aerts4, G. Andria5, T.M. Cox6, M. M.F. Browning1,2,3,4, J. Kennedy2, J. Falardeau2, S.A. Slaugenhaupt2,3. 1Harvard Giralt7, M.K. Mistry8, A. Tylki-Szymanska9. 1Div & Prog Human Genetics, Cinn Medical School Genetics Training Program, Boston, MA; 2Center for Human Ge- Childrens Hosp Med Ctr, Cincinnati, OH; 2Dept. NeurologyNew York University netic Research, Massachusetts General Hospital, Boston, MA; 3Harvard Medical School of Medicine, New York, New York; 3Interantional Collaborative Gaucher School, Boston, MA; 4Childrens Hospital Boston, Boston, MA. GroupUniversity Research Foundation for Lysosomal Storage DiseasesCoral BACKGROUND: Mucolipidosis Type IV (MLIV; MIM 252650) is a metabolic Springs, Fl; 4Dept. Medical BiochemistryAcademic Medical Centre, Amsterdam, neurodegenerative disorder characterized by severe neurologic and ophthalmologic The Netherlands; 5Dept. PediatricsFederico University, Naples, Italy; 6Dept. Medi- abnormalities. Classified as a lysosomal storage disorder, it is progressive and cineUniversity of CambridgeAddenbrooke's Hospital, Cambridge, UK; 7Servico de presents during the first year of life with mental retardation, corneal opacities, achlo- Hemat.Hospital Miguel Servet, Zaragoza, Spain; 8Dept. Intern. Med.Yale Universi- rhydria, and delayed milestones. This research group mapped the MLIV gene to ty School of MedicineNew Haven, CT; 9Dept. Metabolic DiseasesThe Children's chromosome 19p13.2-13.2 and identified the mutant gene, MCOLN1, that encodes Memorial Health Institute, Warsaw, Poland. a novel transmembrane protein, mucolipin-1. Studies have shown MLIV patients are Background: Gaucher disease is a chronic and highly heterogeneous lysosomal constitutively achlorhydric and have partially activated parietal cells with a resulting storage disorder. Enzyme therapy with mannose-terminated glucocerebrosidase hypergastrinemia but the etiology of this achlorhydria remains unknown. (Ceredase, Cerezyme) has been available since 1991. In the years since then, the METHODS: In order to begin to dissect cellular pathways in which mucolipin-1 need for therapeutic goals and an evidence-based disease management approach has acts, we utilized Affymetrix microarray technology to identify genes with differen- become apparent. The cumulative data and clinical experience now exist to allow for tial expression in MLIV. RNA from eight fibroblast cell lines, four MLIV and four the development of such goals and management approach. MethodsIn 2003, an in- control, were hybridized to Affymetrix HG-U133 Plus 2.0 GeneChips at the Harvard ternational panel of physicians with clinical and scientific expertise in GD met to re- Partners Center for Genetics and Genomics Gene Chip Microarray Facility. These view the medical literature and their collective clinical experience in order to chips permit simultaneous analysis of 47,000 transcripts and variants, including develop evidence-based therapeutic goals. ResultsFor each potentially affected or- 38,500 well-characterized human genes. Data were analyzed using the Affymetrix gan compartment, quantitative goals and expected timeframes for achieving those comparison analysis algorithms. goals were developed. Goals for bone disease, anemia, thrombocytopenia, RESULTS: The analysis showed that COPA, which encodes α-COP, a key compo- hepatomegaly, splenomegaly, growth retardation, pulmonary disease, and function- nent of the heptameric coat protein complex I (COPI), was upregulated in MLIV fi- al health were established.Clinical ApplicationTherapeutic goals are a central com- broblasts. COPA was upregulated in all four MLIV lines, with an average fold ponent of a disease management approach for GD which also includes evaluation change of 3.9 (p <0.0003). The α-COP protein is a key component for the assembly and monitoring guidelines developed by the International Collaborative Gaucher of a coat vesicle for transport of proteins and lipids along the secretory pathway for Group (ICGG) and Patient Case Reports from the ICGG Gaucher Registry. Togeth- eukaryotic cells. The 25 amino acid peptide sequence of the N-terminus of α-COP er, these components can be applied to the management of individual patients encodes xenin, [Met-Leu-Thr-Lys-Phe-Glu-Thr-Lys-Ser-Ala-Arg-Val-Gly-Leu- throughout the course of their treatment. The high degree of heterogeneity of GD ne- Ser-Phe-His-Pro-Lys-Arg-Pro-Trp-Ile-Leu-OH] a biomarker that has several bio- cessitates an individualized approach to therapy and a systematic approach of the in- logical activities including inhibition of pentagastrin-stimulated secretion of acid. itial evaluation and subsequent monitoring of patients with GD. The objective of CONCLUSIONS: Microarray analysis of MLIV and control fibroblasts shows an such a disease management approach is to achieve all therapeutic goals within the upregulation of COPA and its encoded protein, α-COP, and presumptively its N-ter- expected timeframes, and once achieved, to maintain these goals over time. The goal minus bioactive form, xenin. We hypothesize that this final pathway leading to in- of this approach is to optimize short- and long-term health outcomes for patients creased xenin may play a role in the achlorhydria observed in MLIV patients as well with GD. In this session, the newly-developed therapeutic goals will be presented in as a potential biomarker providing surveillance in this disease. Future work in eval- the context of such a disease management approach. uating α-COP and xenin expression, and how the absence of mucolipin-1 alters this secretory pathway will provide insight into the pathogenesis of mucolipidosis type IV. A8
  2. 2. PLATFORM PRESENTATIONS BIOCHEMICAL GENETICS 29 30 Lysosomal Cystine Enhanced Programmed Cell Death: Role of PKCδ. M. Park, Clinical, biochemical, and molecular characterization of 79 patients with hear- J. Thoene. Hayward Genetics Center, Tulane University Health Sciences Center, ing loss and multisystemic mitochondrial disease. L.-J.C. Wong1, C-H. Hsu1, H. New Orleans, LA. Kwon1, R.-K. Bai1, C.-L. Perng1, H.-M. Chang2, Y. Zhang3, P. Dai1, A. Gropman4, Cystinosis is characterized by increased lysosomal cystine due to defective trans- F. Scaglia5. 1Inst Molecular Human Genetics, Georgetown Univ Medical Ctr, Wash- port of cystine from the lysosomal compartment. Our previous research has shown ington, DC; 2Vitagenomics, Taipei, Taiwan; 3Lombardi Cancer Ctr, Georgetown that cystinotic fibroblasts and renal proximal tubule epithelial (RPTE) cells have a Univ Medical Ctr, Washington, DC; 4Dept Pediatrics, Georgetown Univ Medical two-fold or more increase in the rate of apoptosis over their normal counterparts Ctr, Washington, DC; 5Dept Mol Hum Genetics, Baylor College of Medicine, Hou- when treated with TNF-α, UV light or anti-Fas antibodies. This increase is alleviated ston, TX. in cystinotic fibroblasts and RPTE cells by pre-treatment with mercaptoeth- Mitochondrial respiratory chain disorders are clinically and genetically heteroge- anolamine, depleting lysosomal cystine. Pre-treatment of normal cells with cystine neous, with central nervous system, skeletal and cardiac muscle being the most sus- dimethylester (CDME), which loads lysosomes with cystine, increases the rate of ceptible tissues. Sensorineural hearing loss is a common clinical feature observed in apoptosis in normal fibroblasts and RPTE cells to cystinotic levels (Park et al, 2002, mitochondrial disorders. The most well studied mutation is the one affecting the 12s Park et al, 2004). We hypothesize that induction of apoptosis in cystinotic cells caus- rRNA gene (A1555G) causing the nonsyndromic hypersensitivity to the ototoxic ef- es a release of cystine into the cytosol that leads to the observed increase in pro- fects to aminoglycosides. Multisystemic mitochondrial cytopathies such as MELAS grammed cell death (PCD) by forming a disulfide bond with one or more apoptotic (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like epi- proteins. To determine whether lysosomal permeabilization in PCD occurs prior to sodes), MERRF (myoclonic epilepsy and ragged red fibers), and Kearns-Sayre syn- irreversible mitochondrial involvement with cytochrome C release, we examined lo- drome (KSS) have been associated with sensorineural hearing loss. However, a calization of both cathepsin B, a lysosomal protease, and cytochrome C, a mitochon- systematic investigation of the molecular defects of syndromic mitochondrial hear- drial protein, using simultaneous immunohistochemistry. In both normal and ing loss has never been conducted. We have previously found that the frequency of cystinotic cells treated with TNF-α, cathepsin B has lysosomal localization at 0 h and sensorineural hearing loss in a cohort of patients with mitochondrial disease was a diffuse cytosolic localization after 4 h exposure. Mitochondrial cytochrome C 21%. In this study, we evaluated the molecular defects in 79 patients diagnosed with however, still shows a punctate localization pattern at 4 h, and does not display a dif- syndromic mitochondrial hearing loss by using the modified adult criteria. Deleteri- fuse pattern until 12 h. These results confirm that lysosomes become permeabilized ous mtDNA mutations and/or mtDNA depletion/multiple deletions were identified early in the apoptotic pathway, upstream of mitochondrial membrane transition. PK- in 28 patients (35%) with syndromic mitochondrial hearing loss. Pathogenic muta- Cδ, a proapoptotic protein kinase, is involved in the process of lysosomal cystine en- tions in mitochondrial tRNA and rRNA genes accounted for approximately 50% of hanced cell death. It has recently been found that cystine increases the activity of the observed mtDNA defects. The other 50% was associated with multiple mtDNA PKCδ in vitro (Chu et al, 2003). We demonstrate that PKCδ forms disulfide bonds deletions, mtDNA depletion, and mtDNA proliferation, possibly secondary to nucle- with 35S-cystine released from lysosomes in cultured cells during apoptosis. The rate ar gene defects. In addition to known pathogenic mutations, TTGE analysis of the of PCD in cystinotic fibroblasts treated with TNF-α is significantly decreased entire mitochondrial genome, identified several novel mutations in mitochondrial (78.2%±2.9 to 51.1%±2.9) after exposure to PKCδ siRNA (p<0.05). This significant tRNA and mRNA genes believed to be pathogenic. In summary, this study repre- decrease is not seen in normal fibroblasts which show a decrease of 40.2%±12.5 to sents the most comprehensive molecular analysis of the mitochondrial genome in 32.7%±2.5 (p>0.05). The activity of PKCδ immunoprecipitated from cystinotic cells patients with syndromic mitochondrial hearing loss. These results would ultimately after TNFα exposure had a 2.5-fold greater activity than PKCδ immunoprecipitated help in understanding the molecular pathomechanism leading to hearing loss in mi- from normal cells. PKCδ in cystinotic cells also coimmunoprecipitates with a nearly tochondrial cytopathies. 2-fold greater amount of lamin B, a PKCδ substrate, than PKCδ from normal conrol cells after exposure to TNFα. This indicates that cystine released from the lysosome causes increased activity of PKCδ in intact cells, which may play a role in the ob- served increase in apoptosis. 31 32 Analysis of homocysteine and methylmalonic acid in blood spots by tandem Autism spectrum manifestations in cerebral folate deficiency. F. Scaglia1, S. mass spectrometry as a second tier newborn screening test for homocystinuria Peters2, K. Hyland3, T. Bottiglieri4, R. Hopkin5, E. Peach5, B. Roa1, C. Bacino1, P. and methylmalonic acidemias. C.D. Cuthbert, M.J. Magera, S.H. Hahn, P. Rinal- Moretti1. 1Dept Molecular & Human Gen, Baylor Col Medicine, Houston, TX; do, S. Tortorelli, D. Matern. Biochemical Genetics Laboratory, Mayo Clinic College 2Dept Pediatrics, Baylor Col Medicine, Houston, TX; 3Horizon Molecular Medi- of Medicine, Rochester, MN. cine, Atlanta, GA; 4Inst Metab Disease, Dallas, TX; 5Dept Human Gen, Cincinnati, Background: Newborn screening for homocystinuria (HCYuria), methylmalonic OH. acidemias (MMAs) and propionic acidemia (PA) is currently mandated in 35 (HCY- Intracellular folates participate in essential one-carbon transfer reactions. Progres- uria) and 21 (MMAs, PA) states. The markers for these disorders are methionine sive neurological dysfunction has been described in adults and children with an iso- (Met) for HCYuria and propionylcarnitine (C3AC) for MMAs and PA. While C3AC lated deficiency of folate in the central nervous system. We studied the clinical and is usually significantly elevated in PA, neither Met nor C3AC are specific for these biochemical features of 5 girls and 2 boys with cerebral folate deficiency (CFD). All diseases and conservative cut off values result in false-positive results requiring un- patients had low cerebrospinal fluid (CSF) levels of 5-methyltetrahydrofolate, the necessary follow up investigations. biologically active form of folates in CSF and blood. Red blood cell folate, serum Objective: To minimize false-positive results associated with Met and C3AC eleva- folate, and total plasma homocysteine were normal. All children exhibited psycho- tions, we developed a method for the determination of methylmalonic acid (MMA) motor retardation and seizures with variable response to folinic acid. Two females and homocysteine (HCY) in dried blood spots (DBS) by liquid-chromatography tan- had manifestations evocative of Angelman syndrome but normal DNA methylation dem mass spectrometry (LC-MS/MS). studies and UBE3A mutation analysis. A third female exhibited features of Rett syn- Methods: 3/16-Inch blood spots were punched out of DBS of 20 controls (C3AC: drome in the absence of MECP2 mutations or deletions. Four of these patients dem- <5.25 µM; Met: <60 µM), 9 newborns with elevated C3AC (range: 5.8-16.95 µM), onstrated considerable repetitive behaviors, decreased eye gaze, and communication 3 newborns with elevated Met (range: 67-112 µM), and 1 patient with an elevated deficits that warranted a formal behavioral evaluation. Using the Autism Diagnostic C3AC (9.45 µM) with a subsequently proven diagnosis of methylmalonic acidemia Interview-Revised, three patients met criteria for autism or autism spectrum disor- due to cobalamin (Cbl) C deficiency. The DBS were eluted by vortexing in an aque- ders. Regardless of their overall cognitive abilities, these children demonstrated def- ous solution containing dithiothreitol and deuterium labeled MMA and HCY as in- icits in communication and socialization that mirror those observed in children with ternal standards (IS). Samples were extracted using a strong anion exchange column idiopathic autism. These findings demonstrate that children with mental retardation, and both the flow-through and the eluate were retained for analysis. The eluate was autism, and clinical features of Angelman syndrome or Rett syndrome may have ab- dried, butylated and analyzed by positive electrospray LC-MS/MS using selected re- normalities of CSF folate, expanding the clinical spectrum of CFD. Future studies action monitoring of transitions m/z 231 to 119 and m/z 234 to 122 for MMA and its will determine the general relevance and frequency of these findings and elucidate IS respectively. The flow-through was mixed with acetonitrile and HCY and its IS the molecular and biochemical mechanisms of reduced CSF folate in these patients. were determined by the transitions m/z 136 to 90 and m/z 140 to 94 respectively. Quantitation was performed using MMA and HCY spiked calibrator DBS spanning a range of 0-250 µM. Analysis time was 5 minutes. Results: MMA and HCY were undetectable in all samples except the DBS of the patient with Cbl C deficiency (MMA: 51 µM; HCY 180 µM). Conclusion: This new LC-MS/MS based method for the determination of both MMA and HCY in DBS has the potential to reduce the number of false positive cas- es due to elevated concentrations of Met and C3AC detected in the primary newborn screening. A9