Morbus Fabry Arndt Rolfs M D Email arndt

Morbus Fabry Arndt Rolfs, M. D. Email: arndt. rolfs@med. uni-rostock. de Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Disclosure The speaker disclocures the following financial support from industry in the last years: o Unrestricted, educational grants from Shire HGT, Genzyme, Actelion, Biomarin, Amicus o Travelling support and honorarium for this talk o Scientific grants from Centogene AG/Germany SWITCH study was financially supported by Shire for the first 12 months; since then the study is exclusively paid by a scientific grants from the University of Rostock Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Lysosomal disease · Nearly 60 different types of lysosomal disease · Most characterized by prominent lysosomal storage (but primary storage substrates vary) · Two-thirds affect brain · Disease cascades poorly understood, and few treatments available Lysosomal enzyme defects Defects in other proteins Pompe disease (glycogenosis) Niemann-Pick type C disease Tay-Sachs, Sandhoff diseases Mucolipidosis II/III Niemann-Pick A/B disease Sialidosis and Galactosialidosis GM 1 gangliosidosis Mucolipidosis IV Gaucher disease Juvenile Batten disease (CLN 3) Fabry disease Cystinosis All MPS diseases Sialic Acid Storage disease Infantile and Late Infantile Batten diseases (CLN 1 and CLN 2) Salla disease -Mannosidosis Danon disease Fucosidosis Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

QUIZ How common are lysosomal diseases? a. 1 in 5000 live births b. 1 in 50, 000 live births c. 1 in 100, 000 live births Answer: a. Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

QUIZ With what frequency are children born world-wide with a lysosomal disease? a. Every 30 minutes b. Every 2 hours c. Once a day Answer: a. Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Fabry disease • Fabry disease is an X-linked lysosomal storage disorder- caused by deficiency of -galactosidase A • Enzyme deficiency leads to accumulation of globotriaosylceramide (Gb 3) • Frequency is estimated in the general population for 1 : 4. 500 and 1: 12, 000 • 0. 5 to 1. 0% of all young stroke patients are suffering from Fabry disease Brady RO et al. N Engl J Med 1967; 276: 1163 7. Meikle PJ et al. JAMA 1999; 281: 249 54 Lappa et al. , JAMA 2006 Rolfs et al, LANCET, 2005 Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Fabry pathology Accumulation of Gb 3 damages o Epithelial cells (e. g. renal epithelium, airway epithelium) o Myocardial cells o Dorsal root ganglia o Neuronal cells o Cells of the autonomic nervous system o Endothelial, perithelial and smooth muscle cells of the vascular system Brady RO et al. N Engl J Med 1967; 276: 1163 7. de. Veber GA et al. Ann Neurol 1992; 31: 409 15. Kahn P. J Neurol Neurosurg Psychiatry 1973; 36: 1053 62. Kaye EM et al. Ann Neurol 1988; 23: 505 9 Courtesy of Dr. Jansen/Essen Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Fabry disease, X-linked genetic, multi-organ disorder Globotriaosylceramide (Gb-3) Lactosylceramide + Galactose -galactosidase A Eyes Brain Skin Kidney Heart Peripheral nervous system Cellular Gb-3 storage TIME Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Screening results in risk populations 9 Patient number Male Fabry Female Fabry Unclear females sum stroke < 55 yrs 14, 267 57 (0. 4%) 91 (0, 6%) 34 (0, 2%) 1, 2%*** proteinuria 4, 637 11 (0. 2%) 15 (0, 3%) 6 (0, 1%) 0, 6% dialysis 4, 838 7 (0, 1%) 11 (0, 2%) 18 (0, 3%) 0, 6% cardiomyopathy 1, 326 3 (0, 2%) 4 (0, 3%) 0, 7% ***p< 0. 01; all patients have been sequenced for the coding GLA stretch and the exon-intron boundaries; samples with mutations/VUS have been tested for enzyme activity and lysio-Gb 3 Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Prevalence of Fabry in Stroke Populations § Rolfs et al Lancet 2005 (Germany); Stroke 2013 prevalence in cryptogenic stroke in 721 (males and females; 18 -55 yrs age) Ø 4. 9% of males (mean age 38. 4 yrs) Ø 2. 4% of females (mean age 40. 3 yrs) Ø Ø increased dolichoectasia, periventricular white matter lesions Ø Fabry symptoms: sensory neuropathic pain, proteinuria, corneal whorls § Brouns et al Stroke 2010 (Belgium) Ø screened consecutive 1000 (18 -60 yrs age; 54. 7% males) with stroke, TIA, intracranial hemorrhage Ø unexplained white matter lesions Ø vertebrobasilar dolichoectasia 1% confirmed by enzyme/molecular analysis Ø Ø § Baptista et al Stroke 2010 (Portugal) Ø (one year experience) first stroke (18 -55 yrs age) n=493 (61% males) Ø 12 (2. 4%) with GLA mutations o 5 with cardiac source, 4 ischemic, 2 hemorrhagic, 1 thrombosis Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Rolfs et al. , 2005 Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

The sifap Study – Stroke in Young Fabry Patients 12 v Stroke in Young Fabry Patients v Prospective, multicenter European study v 5, 024 young stroke patients v 15 European countries, 47 study centers v Age 18 -55 years v Primary aim: Prevalence of Fabry disease in young stroke patients v Secondary aim: Description of patterns of stroke in young patients v Detailed characterization of clinical, laboratory and radiological data v All patients tested for Fabry disease genetically Rolfs et al Lancet 2005; Rolfs et al. Stroke 2013; Fazekas et al. Neurology 2013 Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Fabry Disease and Type of CVE 13 Cross-classified table classcve_1 classification of cve 1, 00 TIA fabry_diagnosis of fabry Gesamt 1, 00 yes Anzahl 3, 00 no fabry Anzahl % % Anzahl % 2, 00 primary hemorrhage 3, 00 ischemic stroke Total 4, 00 other 4 2 19 0 25 16, 0% 8, 0% 76, 0% 0, 0% 1062 269 3364 68 4763 22, 3% 5, 6% 70, 6% 1, 4% 100, 0% 1066 271 3383 68 4788 22, 3% 5, 7% 70, 7% 1, 4% 100, 0% p=0. 743 for Fishers exact test (not adjusted for centre heterogeneity, which is not possible because of small number of cases) • 27 Fabry patients • 0. 5% of all sifap patients (5005 without Fabry disease) • 18 uncertain cases not included in analysis Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Fabry Disease and Gender 14 Cross-classified table SEX Sex 1 male fabry_diagnosis of fabry 1, 00 yes Anzahl 3, 00 no fabry Anzahl Gesamt % % Anzahl % Total 2 female 11 16 27 40, 7% 59, 3% 100, 0% 2947 2031 4978 59, 2% 40, 8% 100, 0% 2958 2047 5005 59, 1% 40, 9% 100, 0% p=0. 075 (Fisher’s exact test) Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Family History of Cardiac Disease 15 Cross-classified table FAMHIST 1 Is there any family history for cardiac diseases 0 no fabry_diagnosis of fabry Gesamt 1, 00 yes Anzahl 3, 00 no fabry Anzahl % % Anzahl % p=0. 323 in fishers exact test Total 1 yes 18 8 26 69, 2% 30, 8% 100, 0% 2771 1932 4703 58, 9% 41, 1% 100, 0% 2789 1940 4729 59, 0% 41, 0% 100, 0% p=0. 323 Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Family History of Renal Disease 16 Cross-classified table Total FAMHIST 2 Is there any family history for renal diseases 0 no fabry_diagnosis of fabry Gesamt 1, 00 yes Anzahl 3, 00 no fabry Anzahl % % Anzahl % 1 yes 2 unknown 21 5 1 27 77, 8% 18, 5% 3, 7% 100, 0% 4100 516 362 4978 82, 4% 10, 4% 7, 3% 100, 0% 4121 521 363 5005 82, 3% 10, 4% 7, 3% 100, 0% p=0. 341 Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Family History of CVE 17 Cross-classified table Total FAMHIST 3 Is there any family history for CVE diseases 0 no fabry_diagnosis of fabry Gesamt 1, 00 yes Anzahl 3, 00 no fabry Anzahl % % Anzahl % 1 yes 18 7 25 72, 0% 28, 0% 100, 0% 2969 1748 4717 62, 9% 37, 1% 100, 0% 2987 1755 4742 63, 0% 37, 0% 100, 0% p=0. 411 Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Prior MIs 18 Cross-classified table Total MEDHIST 5 Prior myocardial infarctions 0 no fabry_diagnosis of fabry Gesamt 1, 00 yes Anzahl 3, 00 no fabry Anzahl % % Anzahl % 1 yes 26 1 27 96, 3% 3, 7% 100, 0% 4799 153 4952 96, 9% 3, 1% 100, 0% 4825 154 4979 96, 9% 3, 1% 100, 0% p=0. 573 Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Age at symptom onset No Fabry Uncertain Mean 44. 7 41. 5 43. 5 Median 47. 0 45. 0 44. 0 Std. Deviation 8. 2 10. 5 8. 1 P<0. 05 Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Total protein in urine (mg/dl) No Fabry Uncertain Mean 38. 5 190. 3 61. 0 Median 6. 7 9. 0 8. 1 Std. Deviation 470. 2 928. 2 14. 5 P<0, 05 Beck Depression Inventory – BDI II No Fabry Uncertain Mean 7. 1 8. 4 6. 5 Median 5. 0 7. 0 5. 0 Std. Deviation 7. 7 7. 9 4. 6 P<0, 05 Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

K. Sims et al. , 2009; Stroke in Fabry patients Percentages of patients experiencing their first stroke during 6 age categories are shown. Data for males are in dark gray bars and data for females are in light gray bars, with the number of patients in each age category shown above each bar. All data are from patients not treated with ERT at the time of their first stroke. Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Medical history of risk factors in Fabry with stroke Arrhythmia was more prevalent in male stroke patients. Hypertension was more strongly associated with stroke in female patients. Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty Sims et al 2009

Central Nervous System Pathology LARGE VESSEL n Dolichoectasia brainstem vulnerability SMALL VESSEL n n § n Susceptibility to occlusive vascular thrombosis increased endothelial prothrombotic factors Changes in regional CBF [r. CBF] cellular metabolic failure Increased ROS metabolic stress Chronic alteration in nitric oxide pathway [NO] altered vascular tone & reactivity WHITE MATTER § § Perfusion changes, fluid shifts Decreased regional cerebral glucose metabolism GRAY MATTER § § Decreased NAA – cortical & subcortical neurons Calcification (pulvinar sign) Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Structural lesions in the MRI in Fabry disease • 50% of all patients ( n=50, mean age 45 yrs ) demonstrating WML • all patients > 54 yrs with microvascular changes • No patient < 26 yrs with structural lesions in the MRI • About 40 % of patients with lesions suffering from neurological symptoms Crutchfield et al. , Neurology 1998 • 33% of all pts with Fabry with significant WML Fellgiebel et al. , Neurology 2005; Fellgiebel et al. , Lancet Neurol 2006 Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Fabry patients show increased diameter of cerebral vessels A. Fellgiebel et al, 2011; 2013 95% CI = 95% confidence Interval; post. = posterior cerebral artery; med. = middle cerebral artery; ant. = anterior cerebral artery; carot. = Carotid artery; comp. = Composite z-score (mean z over all arteries) Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Fabry – conjunctival and retinal angiopathy Ampulla-like conjunctival vessels (98% of all male cases) Dilatation and tortoous retinal vessels (76%) Normal Courtesy of Prof. Dr. R. Guthoff, Rostock Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

D 313 Y – mutation or SNP ? How to classify ? Multifocal White Matter Lesions Associated with the D 313 Y Mutation of the a-Galactosidase A Gene (PLOSone, 2013) Malte Lenders 1. , Thomas Duning 2. , Michael Schelleckes 1, Boris Schmitz 1, 3, Sonja Stander 4, Arndt Rolfs 5, Stefan-Martin Brand 3, Eva Brand 1* Muenster/Rostock, Germany Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Biomarker lyso-Gb 3: best but not yet the ideal marker (sensitivity: m 92. 4%; f 89. 6%) Pathological cut-off: 0. 9 ng/ml plasma (95% confidence interval) Median lyso-Gb 3 values were higher in males. Specificity (0. 5) and Sensitivity (1. 0) was nearly the same for both genders. "Non-pathological" variants of the GLA gene were restricted to a few genotypes here: S 126 G (pink dots), A 143 T (blue dots), D 313 Y (green dots) J. Lukas, 2013 Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty 28

Biomarker Lyso-Gb 3 is a reliable indicator for severe mutations in Fabry patients 29 Males females In-vitro enzyme activity < 1%: severe 1 -20%: moderate >20%: mild • Severe mutations are significantly related to the classic phenotype in FD • Lyso-Gb 3 reflects disease severity as an in-vivo parameter Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Mild mutations are located on the molecule surface 30 - Mutations D 83 N, D 175 N, S 102 L, R 252 T, D 313 Y, T 385 A, F 396 Y, E 398 A display only mild effects on GLA activity. S 126 G I 219 T R 220 Q Provided by Prof. A. Markoff, University of Muenster - H-bond disturbances like in the shown mutations are predicted to display only mild effects on enzyme performance Lukas et al. , 2015 Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

D 313 Y (Score 5) mutation and Gb 3 - Fabry or „Gb 3 emia“ ? PATIENT exon mutation sex P 0037 c 937 G>T [D 313 Y] m P 0431 c 937 G>T [D 313 Y] P 0558 ALPHAGALACT Gb 3 blood µg/ml 31 Gb 3 IHC skin Gb 3 EM skin 10, 2 3, 1 f 27 3, 9 negative c 937 G>T [D 313 Y] f 21 3, 0 negative P 0607 c 937 G>T [D 313 Y] f 14 2, 2 P 0691 c 937 G>T [D 313 Y] m 3, 6 6, 4 negative P 0649 c 937 G>T [D 313 Y] f 22 2, 7 P 0839 c 937 G>T [D 313 Y] m 12, 7 5, 1 unclear P 0865 c 937 G>T [D 313 Y] f 43 3, 6 P 1236 c 937 G>T [D 313 Y] m 17 P 1515 c 937 G>T [D 313 Y] m 15 5, 8 P 3102 c 937 G>T [D 313 Y] m 19 0, 9 P 2143 c 937 G>T [D 313 Y] m 3, 4 P 4024 c 937 G>T [D 313 Y] f 11, 8 P 2704 c 937 G>T [D 313 Y] m 36 P 2871 c 937 G>T [D 313 Y] f 29 P 2918 c 937 G>T [D 313 Y] m 2, 4 P 3607 c 937 G>T [D 313 Y] f 48 total Gb 3_24 91, 32 21, 18 negative 120, 00 24, 40 negative 77, 00 16, 10 269, 11 58, 17 positive 49, 00 11, 60 negative 130, 89 33, 15 negative 228, 77 45, 59 4, 6 negative 317, 19 70, 63 3, 1 negative 97, 76 19, 58 7, 5 negative 67, 00 22, 30 371, 09 79, 60 Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Two patients with D 313 Y – is that Fabry disease ? 32 Pts. AW, 28 yrs, male, south Germany: severe cardiomyopathy, hypohidrosis, angiokeratoma, pain in childhood, no Cornea verticillata; a-Gal activity: 5. 1* (male Fabry pts. 0 – 6. 4 n. Mol MU/mg protein; normal 33 -109); Gb 3 blood 5. 3 µg/ml (normal < 4. 0 µg/ml), lyso-Gb 3 2. 8 (normal < 1. 1 ng/ml) Pts. UK, 41 yrs, male, northern Poland: severe proteinuria, hypohidrosis, stroke and 4 TIAs in last 5 years, mild cardiomyopathy, no angiokeratoma, no Cornea verticillata; a-Gal activity: 7. 2** (n. Mol MU/mg protein); Gb 3 blood 4. 9 µg/ml (normal < 4. 0 µg/ml), lyso-Gb 3 1. 7 (normal < 1. 1 ng/ml) - MLPA was normal in both cases Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Skin: Gb 3 immunohistochemistry and electron microscopy pat AW - D 313 Y pat UK - D 313 Y Gb 3 immunhohistocehmistry Fabry ctlr. A 156 V Electron micrsocopy EM: typical inclusions with irregular parallel lamellae, characteristic Zebra bodies (Prof. Jonas, Rostock) Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Lyso-Gb 3 of patients on agalsidase alpha (wo back-switch) males females Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Lyso-Gb 3 of patients on agalsidase alpha (wo back-switch) Males (n= 38, 36, 27 with regard to time points) Females (n=14, 13 , 6 with regard to time points) Males: significant decrease over time in lyso gb 3 levels (p=0. 002) (n=137 measures / 38 patients, random intercept models); Females significant decrease over time in lyso gb 3 levels ( p=0. 044) (n=14 patients / 46 measures) Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Is time on A. alpha related to lyso-Gb 3 values? (only values under A. alpha) Males Females Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

CARDIAC PARAMETERS (agalsidase alfa only) 37 Switch V 1 V 2 V 3 Filling impairment % (n) Valvular lesions % (n) Late enhancement % (n) ECG changes % (n) NYHA classification % (n) 26, 3% (15) 26, 4% (19) 30, 4% (21) 24, 2% (8) 28, 6% (18) 24, 3% (18) 29, 4% (20) 15, 2% (5) 8, 1% (5) 50% (25) 51, 3% (40) 3, 7% (2) 59, 4% (41) 3, 0% (1) 43, 3% (13) • NYHA I 22, 1% (15) 18, 8% (16) 19, 2% (15) 11, 1% (4) • NYHA II 11, 8% (8) 16, 5% (14) 15, 4% (12) 13, 9% (5) • NYHA III 4, 4% (3) 5, 9% (5) 10, 3% (8) 0 • NYHA IV 0 0 Hypertension % (n) Pacemaker % (n) 34, 7% (25) 30, 2% (26) 33, 8% (27) 22, 2% (8) 6, 5% (5) 11, 0% (10) 13, 4% (11) 11, 1% (4) Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Creatinine clearance MDRD P=0. 313 (Replagal vs. Fabrazyme)* * adjusted for sex, age, time point and interaction between medication and time point 58 72 73 4 10 22 20 -- -- -- 78 66 53 34 Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

GFR creatinine clearance in ml/min*1. 73 m 2 P=0. 183 (Replagal vs. Fabrazyme)* * adjusted for sex, age, time point and interaction between medication and time point 49 68 57 4 8 19 17 -- -- -- 61 49 33 18 Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Total protein in urine (mg/dl) P=0. 347 (Replagal vs. Fabrazyme)* * adjusted for sex, age, time point and interaction between medication and time point 49 61 58 4 12 22 17 -- -- -- 67 50 36 19 Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Total albumin in urine (mg/dl) P=0. 539 (Replagal vs. Fabrazyme)* * adjusted for sex, age, time point and interaction between medication and time point 37 56 54 4 12 21 17 -- -- -- 67 54 40 23 Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Mainz Severity Score Index P=0. 080 (Replagal vs. Fabrazyme)* * adjusted for sex, age, time point and interaction between medication and time point 55 68 74 4 13 23 18 -- -- -- 86 71 53 35 Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty

Tip of the iceberg model in Fabry Thx indication Classical phenotype (10%) Oligo-/monosymptomatic phenotype +++ Score 10 ++ + Late-onset, monosymptomatic phenotype Clin Score (+) 0 Score 1 Albrecht-Kossel-Institute for Neuroregeneration (AKos) University of Rostock – Medical Faculty AOO lyso. Gb 3