Screening for Fabry disease using dried blood spots in clinically relevant patient populations over six years

Abstracts / Molecular Genetics and Metabolism 117 (2016) S14–S124

Mucopolysaccharidosis type III is a group of four enzyme deficiencies leading to tissue accumulation of heparan sulfate. Central nervous system disease is prominent with initial normal development then cognitive decline, hyperactivity, and death. Somatic findings are modest. Over one year (0, 6, and 12 months), we prospectively assessed disease progression in MPS IIIA and IIIB subjects N2 years old in order to define suitable outcomes for upcoming gene transfer trials. Fifteen IIIA (M = 9, F = 6; age 5.0 ± 1.9 years) and ten IIIB subjects (M = 8, F = 2; age 8.7 ± 3.2 years) were enrolled. Eighteen have completed the 12 month visit (with 2 more expected), and five withdrew, including one due to disease progression and two due to an opportunity for a therapeutic trial elsewhere. A variety of tests that were initially included proved unreliable in this population and were removed from the protocol, including timed motor function tests and the Child Behavior Checklist. Cognitive function (assessed using the Leiter and Mullen scales) maximized at the 2.5-3.0 year old age level and showed agerelated decline, as did parental report of adaptive behavior (Vineland). Analysis of brain volumetrics by MRI is ongoing, but MRI volumes of liver (mean, 2.2X normal volume) and spleen (mean, 1.9X normal volume) were increased. Urine glycosaminoglycan levels decline steeply with age, approaching normal in older subjects, which should be considered when interpreting efficacy in future trials. Similarly, safety endpoints should take into account baseline elevations in CSF protein (which rose with age) and of serum AST and ALT. These results suggest that cognitive and reported behavior may be suitable functional outcomes for a trial, and have helped to guide the design of our AAV-mediated gene transfer trial. doi:10.1016/j.ymgme.2015.12.467

310 Anti-α-galactosidase A antibodies and serum-mediated inhibition in Fabry disease Takahiro Tsukimuraa, Futoshi Shibasakib, Masashi Shigenagaa, Tadayasu Togawaa, Hitoshi Sakurabaa, aMeiji Pharmaceutical University, Kiyose, Japan, bTokyo Metropolitan Institute of Medical Science, Setagaya-ku, Japan Enzyme replacement therapy (ERT) with recombinant α-galactosidase A (GLA) have provided Fabry disease patients with demonstrable benefits. However, recurrent injections of recombinant GLAs sometimes induce production of antibodies against the GLA proteins in patients with Fabry disease. In this study, we measured titer levels of anti-GLA antibodies in serum from Fabry patients and compared the results with the degree of the serum-mediated inhibition, and their phenotype and genotype. We used serum samples from 29 Fabry patients received ERT with agalsidase alfa (Aga-A), agalsidase beta (Aga-B) or the both. We measured titer levels of the antibodies against Aga-A and Aga-B by ELISA and immunochromatography-based assaying. Serum-mediated inhibition was examined by an in vitro GLA inhibition assaying. The patients exhibited the same level of antibodies against both Aga-A and Aga-B, regardless of the species of recombinant GLA used for ERT. Most of the patients who showed immunopositive reaction exhibited classic Fabry phenotype and harbored gene mutations affecting biosynthesis of GLA. The residual GLA activities after incubation with serum in the immune positive group, false positive one, negative one, and normal controls were 12 ± 16, 46 ± 5, 68 ± 5, and 60 ± 5%, respectively. The titer levels of antibodies and the degree of serum-mediated inhibition were almost correlated without an exceptional case that showed strong immunopositive reaction although the serummediated inhibition was weak. It is important to monitor antibodies

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against GLA for patients with Fabry disease during ERT, especially for patients affected with classic Fabry disase having mutations which defect biosynthesis of GLA, because antibodies against GLA often reduce the effect of ERT. doi:10.1016/j.ymgme.2015.12.468

311 Combined analysis of glucosylsphingosine, lyso-sphingomyelin, cholestane-3β,5α,6β-triol, and 7-ketocholesterol in plasma for Gaucher and Niemann-Pick disease types A, B, and C Coleman Turgeon, Dietrich Matern, Piero Rinaldo, Dimitar Gavrilov, Devin Oglesbee, Silvia Tortorelli, Kimiyo Raymond, Mayo Clinic, Rochester, MN, United States The differential diagnosis of patients with isolated splenomegaly or hepatosplenomegaly includes Gaucher (GSD), Niemann-Pick disease type A (NPA), Niemann-Pick disease type B (NPB) or Niemann-Pick disease type C (NPC). We hypothesized that these conditions with similar clinical phenotypes can be differentiated by measuring levels of glucosylsphingosine (GPSY), lyso-sphingomyelin (LSM), Cholestane3β,5α,6β-triol (COT), and 7-ketocholesterol (7-KC) in plasma. Therefore, we developed a method to simultaneously measure plasma glucosylsphingosine (GPSY), lyso-sphingomyelin (LSM), Cholestane3β,5α,6β-triol (COT), and 7-ketocholesterol (7-KC) that is faster than recent approaches. Plasma collected from 119 controls, 14 NPAB, and 14 NPC patients were used for method validation. The method exploited an internal standard (IS) solution containing d7-COT and d7-7-KC to which acetonitrile was added to precipitate plasma proteins. After centrifugation, supernatants were diluted with water and subjected to liquid chromatography-tandem mass spectrometry analysis. The MS/MS was operated in multiple reaction monitoring (MRM) positive mode to detect the underivatized precursor to product species transitions for COT (ammonium adduct), 7-KC, LSM, GPSY and each IS. Total analysis time was 10 minutes. COT, 7-KC and LSM were measured in the validation cohort with 100% clinical sensitivity and specificity. Gaucher patients (N = 5) had markedly elevated concentrations of GPSY (range: 298-1230 nM; controls: b6 nM) and Gaucher carriers (N = 3; GPSY range: 7-9 nM). Preliminary data indicates that the combined analysis of GPSY, LSM, COT, and 7-KC in plasma is useful for the assessment of metabolic disease presenting with hepatosplenomegaly. doi:10.1016/j.ymgme.2015.12.469

312 Screening for Fabry disease using dried blood spots in clinically relevant patient populations over six years Karen L. Tylee, Stephanie Agoreyo, Christine Egerton, Oliver Parkes, Heather J. Church, Manchester Centre for Genomic Medicine, Manchester, United Kingdom The Willink Biochemical Genetics Unit provides screening for Fabry disease in patients from ‘at risk’ cardiac and renal clinical populations. The strategy is to initially measure α-galactosidase A in a DBS, with reduced activity below the established reference range (6.3-47 pmol/punch/hr) followed up by measurement of α-galactosidase A in blood and DNA analysis of the GLA gene. Over 6 years we have screened 5073 patients; 3591 (71%) males and 1470 (29%) females. Subsequent follow up investigations by enzymology and DNA analysis has led to confirmed diagnoses of Fabry disease in 34 patients (~positive rate 1/149). This is an effective strategy for identifying males affected by

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Abstracts / Molecular Genetics and Metabolism 117 (2016) S14–S124

Fabry disease. However, identifying Fabry disease heterozygous status in females remains a challenge with ~ 25-30% of these patients having enzyme activity within the normal reference range. Our data is comparable for both DBS and plasma from known heterozygous females. Our centre has received 171 female referrals for screening of the GLA gene. Of 93 samples with no family history, 68 were found to be normal, 17 were found to have known polymorphisms and 8 were heterozygous for known pathogenic changes. Of the female patients referred with a known family history (n = 77), 49 were identified as heterozygous for the familial mutation and 28 were found to be normal excluding Fabry disease. Many of these patients were referred following diagnosis of Fabry in a male relative from the DBS testing strategy. DBS screening has proved to be an effective strategy for male patients in ‘at risk ‘patient groups, while ~30 % of female patients will be missed by enzymology in isolation. However, many females have been identified by cascade family testing after the diagnosis of a symptomatic male relative and therefore DBS analysis has provided indirect identification of these female patients. doi:10.1016/j.ymgme.2015.12.470

313 Characteristics of patients with MPS II diagnosed at a very young age: Data from the Hunter Outcome Survey (HOS) Anna Tylki-Szymańskaa, Can Ficicioglub, Isabelle Morinc, Agnieszka Jureckad, Barbara K. Burtone, aThe Children’s Memorial Health Institute, Warsaw, Poland, bThe Children’s Hospital of Philadelphia, Philadelphia, PA, United States, cCytel, Inc, Geneva, Switzerland, dShire, Lexington, MA, United States, eAnn & Robert H Lurie Children’s Hospital of Chicago, Northwestern University, Chicago, IL, United States Although the pathological process underlying mucopolysaccharidosis type II (MPS II; Hunter syndrome) starts prenatally, patients usually appear normal at birth, and so early diagnosis remains challenging. Enzyme replacement therapy with idursulfase has been available since 2006. Owing to the progressive nature of the disease, there has been interest in starting treatment as early as possible. This analysis aimed to determine characteristics of patients identified and treated with idursulfase at a very young age, using data from the Hunter Outcome Survey (a global observational registry). As of July 2015, 5.2% of treated patients followed prospectively in HOS had started idursulfase aged ≤18 months (39/757; all male patients). Median age (10th-90th percentiles) of these patients at diagnosis was 0.4 (0.0-1.3) years; the maximum delay in diagnosis was 9.6 months. Median age at ERT initiation was 0.9 (0.1-1.4) years. More than one-third (35.9%; n = 14) were aged b6 months at treatment start; 23.1% (n = 9) were aged 6 to b12 months and 41.0% (n = 16) were aged 12 to ≤18 months. Median age at onset of symptoms was 0.5 (0.0-1.1) years. The main presenting signs and symptoms during early disease course included hernia, hearing loss and hepatosplenomegaly. More than one-quarter (29.7%; n = 11/37) had a negative family history of MPS II. The disease was suspected primarily by medical geneticists, general paediatricians and metabolic disease specialists; however, some diagnoses were made by pulmonologists, ear, nose and throat specialists, and orthopaedic surgeons. Although in the majority of cases a positive family history played a critical role in early diagnosis, almost 30% of patients were diagnosed based on clinical suspicion alone. Further analysis is required to establish whether there are any common features that led to early diagnosis and treatment in these patients. Funding information: Shire sponsors the Hunter Outcome Survey and funds medical writing support. doi:10.1016/j.ymgme.2015.12.471

314 Molecular analysis of 22 patients with mucopolysaccharidosis IVA from Poland, Belarus and Kazakhstan identifies 6 novel GALNS mutations Anna Tylki-Szymańskaa, Agnieszka Różdżyńska-Świątkowskaa, Jolanta Maruchaa, Anna Kulpanovichb, Assel Tulebayevac, Agnieszka Jureckaa, aThe Children’s Memorial Health Institute, Warsaw, Poland, b National Medical Center "Mother and Child", Minsk, Belarus, cScientific Centre of Pediatrics and Child Surgery, Almaty, Kazakhstan Mucopolysaccharidosis type IVA (MPS IVA, Morquio syndrome type A) is an autosomal recessive lysosomal disorder caused by deep deficiency of galactosamine-6-sulfate sulfatase (GALNS), encoded by the GALNS gene. Patients who carry two mutated allele of GALNS gene have a decreased ability to catabolise keratan and chondroitin 6sulfates, which results in their accumulation and leads to connective tissue impairment and consequently to multiorgan clinical outcome. We aimed to analyse the spectrum of mutations in the GALNS gene responsible for the disorder in Poland, Belarus and Kazakhstan. Twenty two patients with MPS IV, in whom diagnosis was confirmed biochemically and enzymatically, were studied. In total, fifteen different disease-causing mutations were identified. Six novel mutations included c.680delT, D183Y, A321G, c.IV13 + 5G N A, c.121-9 T N G, and Q476P. No mutations were identified on 9/44 alleles. Eight patients were homozygous and had either related parents or parents from neighbouring villages. All patients, except one (homozygous for p.R529Q) presented with a severe phenotype. Genotypic data in our multi-ethnic study group, did not allow for prediction of disease severity (phenotype). The analysis failed to identify mutations on 21% of alleles suggesting that the only reliable diagnostic method for Morquio syndrome type A remains enzymatic testing. doi:10.1016/j.ymgme.2015.12.472

315 Pigmentary retinopathy as first manifestation in two cases of Scheie syndrome Geoffrey Urbanskia, Magalie Bartha, Aline Gurya, Stéphanie Lerueza, Christophe Baufretona, Jean-Louis De Bruxa, Xavier Zanlonghib, Dominique Bonneaua, Christian Lavignea, aCentre Hospitalier Universitaire, Angers, France, bClinique ophtalmologique Sourdille, Nantes, France Scheie syndrome (MPS IS) is the most attenuated form of mucopolysaccharidosis type I. Corneal clouding and glaucoma represent the most frequent ocular impairments in MPS IS. Retina could be involved but it is often difficult to highlight because of the corneal clouding. In the literature, pigmentary retinopathy (PR) is described in MPS IS during systematic examinations but data about the clinical course of the PR are poor. We report 2 cases of patients, brother and sister, with MPS IS revealed by hemeralopia due to a PR. The two patients belong to a caucasian non consanguine family. The girl, first diagnosed, developed a hemeralopia at 15 and a severe PR was diagnosed at 26. At 20, she complained with progressive joint stiffness. The diagnosis of MPS IS was done at 27. She had a slightly infiltrated face and a mild aortic and mitral valves thickening. The boy had an aortic and mitral valves replacement at the age of 25 for severe valvular thickening attributed to rheumatic heart disease. PR was diagnosed at 27 but hemeralopia appeared many years before. He developed joint stiffness at 29. None had corneal clouding nor glaucoma. The alpha-L-iduronidase activity in leukocytes was decreased in both patients (0.2 μkat/kg, normal range: 3.4-10.6). Genetic testing of IDUA revealed a double heterozygoty, with a