1. Morbus Fabry Arndt Rolfs, M.D. Email: arndt.rolfs@med.uni-rostock.de

2. Disclosure
The speaker disclocures the following financial support from industry in the last years:
Unrestricted, educational grants from Shire HGT, Genzyme, Actelion, Biomarin, Amicus
Travelling support and honorarium for this talk
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

3. 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
Pompe disease (glycogenosis)
Tay-Sachs, Sandhoff diseases
Niemann-Pick A/B disease
GM1 gangliosidosis
Gaucher disease
Fabry disease
All MPS diseases
Infantile and Late Infantile Batten diseases (CLN1 and CLN2)
-Mannosidosis
Fucosidosis
Defects in other proteins
Niemann-Pick type C disease
Mucolipidosis II/III
Sialidosis and Galactosialidosis
Mucolipidosis IV
Juvenile Batten disease (CLN3)
Cystinosis
Sialic Acid Storage disease
Salla disease
Danon disease

4. 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.

5. 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.

6. Enzyme deficiency leads to accumulation of globotriaosylceramide (Gb3)
Fabry disease
Fabry disease
Fabry disease is an X-linked lysosomal storage disorder- caused by deficiency of -galactosidase A
Enzyme deficiency leads to accumulation of globotriaosylceramide (Gb3)
Frequency is estimated in the general population for 1 : 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

7. Fabry pathology
Fabry pathology
Accumulation of Gb3 damages
Epithelial cells (e.g. renal epithelium, airway epithelium)
Myocardial cells
Dorsal root ganglia
Neuronal cells
Cells of the autonomic nervous system
Endothelial, perithelial and smooth muscle cells of the vascular system
Brady RO et al. N Engl J Med 1967; 276: 11637.
deVeber 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

8. 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

9. Screening results in risk populations
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%)
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-Gb3

10. Prevalence of Fabry in Stroke Populations
Rolfs et al Lancet (Germany); Stroke 2013
prevalence in cryptogenic stroke in 721 (males and females; yrs age)
4.9% of males (mean age 38.4 yrs)
2.4% of females (mean age 40.3 yrs)
increased dolichoectasia, periventricular white matter lesions
Fabry symptoms: sensory neuropathic pain, proteinuria, corneal whorls
Brouns et al Stroke 2010 (Belgium)
screened consecutive 1000 (18-60 yrs age; 54.7% males) with
stroke, TIA, intracranial hemorrhage
unexplained white matter lesions
vertebrobasilar dolichoectasia
1% confirmed by enzyme/molecular analysis
Baptista et al Stroke 2010 (Portugal)
(one year experience) first stroke (18-55 yrs age) n=493 (61% males)
12 (2.4%) with GLA mutations
5 with cardiac source, 4 ischemic, 2 hemorrhagic, 1 thrombosis

11. Rolfs et al., 2005

12. The sifap Study – Stroke in Young Fabry Patients
Prospective, multicenter European study
5,024 young stroke patients
15 European countries, 47 study centers
Age years
Primary aim: Prevalence of Fabry disease in young stroke patients
Secondary aim: Description of patterns of stroke in young patients
Detailed characterization of clinical, laboratory and radiological data
All patients tested for Fabry disease genetically
Rolfs et al Lancet 2005; Rolfs et al. Stroke 2013; Fazekas et al. Neurology 2013

13. Fabry Disease and Type of CVE13
Cross-classified table
classcve_1 classification of cve
Total
1,00 TIA
2,00 primary hemorrhage
3,00 ischemic stroke
4,00
other
fabry_diag diagnosis of fabry
1,00 yes
Anzahl 4 2 19 25 %
16,0%
8,0%
76,0%
0,0%
100,0%
3,00 no fabry
1062
269
3364 68
4763
22,3%
5,6%
70,6%
1,4%
Gesamt
1066
271
3383
4788
5,7%
70,7%
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 13

14. Fabry Disease and Gender14
Cross-classified table
SEX Sex
Total
1 male
2 female
fabry_diag diagnosis of fabry
1,00 yes
Anzahl 11 16 27 %
40,7%
59,3%
100,0%
3,00 no fabry
2947
2031
4978
59,2%
40,8%
Gesamt
2958
2047
5005
59,1%
40,9%
p=0.075 (Fisher’s exact test) 14

15. Family History of Cardiac Disease
Cross-classified table
FAMHIST1 Is there any family history for cardiac diseases
Total
0 no
1 yes
fabry_diag diagnosis of fabry
1,00 yes
Anzahl 18 8 26 %
69,2%
30,8%
100,0%
3,00 no fabry
2771
1932
4703
58,9%
41,1%
Gesamt
2789
1940
4729
59,0%
41,0%
p=0.323 in fishers exact test
p=0.323

16. Family History of Renal Disease16
Cross-classified table
FAMHIST2 Is there any family history for renal diseases
Total
0 no
1 yes
2 unknown
fabry_diag diagnosis of fabry
1,00 yes
Anzahl 21 5 1 27 %
77,8%
18,5%
3,7%
100,0%
3,00 no fabry
4100
516
362
4978
82,4%
10,4%
7,3%
Gesamt
4121
521
363
5005
82,3%
p=0.341

17. Cross-classified table
Family History of CVE 17
Cross-classified table
FAMHIST3 Is there any family history for CVE diseases
Total
0 no
1 yes
fabry_diag diagnosis of fabry
1,00 yes
Anzahl 18 7 25 %
72,0%
28,0%
100,0%
3,00 no fabry
2969
1748
4717
62,9%
37,1%
Gesamt
2987
1755
4742
63,0%
37,0%
p=0.411

18. Cross-classified table
Prior MIs 18
Cross-classified table
MEDHIST5 Prior myocardial infarctions
Total
0 no
1 yes
fabry_diag diagnosis of fabry
1,00 yes
Anzahl 26 1 27 %
96,3%
3,7%
100,0%
3,00 no fabry
4799
153
4952
96,9%
3,1%
Gesamt
4825
154
4979
p=0.573

19. Age at symptom onset Mean 44.7 41.5 43.5 Median 47.0 45.0 44.0
No Fabry
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
@Beate: kannst du bitte noch die n#s für die jeweiligen gruppen angeben

20. Total protein in urine (mg/dl)
No Fabry
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
Fabry
Uncertain
Mean
7.1
8.4
6.5
Median
5.0
7.0
Std. Deviation
7.7
7.9
4.6
P<0,05
Auch bei diesen „neuen“ Variablen zeigen sich höchstwahrscheinlich Probleme mit Fehleinträgen bei Units; es gibt z.B. eine Person mit Maximum mg/dl. Das ist bestimmt nicht sinnvoll, wenn der Median so „niedrig“ ist. Es gab auch negative Werte, die ich durch die Beschränkung der y-Achse im Plot nun einfach abgeschnitten habe. Fraglich, ob man das so (außer Median) schon interpretieren kann.
Ug: wie viele extremewerte waren es denn?

21. 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.

22. 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.
Arrhythmia was more prevalent in male stroke patients.
Hypertension was more strongly associated with stroke in female patients.
Sims et al 2009 22

23. Central Nervous System Pathology
LARGE VESSEL
Dolichoectasia
brainstem vulnerability
SMALL VESSEL
Susceptibility to occlusive vascular thrombosis
increased endothelial prothrombotic factors
Changes in regional CBF [rCBF]
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)

24. 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

25. 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)

26. Fabry – conjunctival and retinal angiopathy
Normal
Ampulla-like conjunctival vessels (98% of all male cases)
Dilatation and tortoous retinal vessels (76%)
Courtesy of Prof. Dr. R. Guthoff, Rostock

27. D313Y – mutation or SNP ? How to classify ?
Multifocal White Matter Lesions Associated with the D313Y Mutation of the a-Galactosidase A Gene (PLOSone, 2013)
Malte Lenders1., Thomas Duning2., Michael Schelleckes1, Boris Schmitz1,3, Sonja Stander4, Arndt Rolfs5,
Stefan-Martin Brand3, Eva Brand1*
Muenster/Rostock, Germany

28. Biomarker lyso-Gb3: best but not yet the ideal marker (sensitivity: m 92.4%; f 89.6%)28
Pathological cut-off: 0.9ng/ml plasma (95% confidence interval)
Median lyso-Gb3 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:
S126G (pink dots), A143T (blue dots), D313Y (green dots)
J. Lukas, 2013

29. Lyso-Gb3 reflects disease severity as an in-vivo parameter
Biomarker Lyso-Gb3 is a reliable indicator for severe mutations in Fabry patients
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-Gb3 reflects disease severity as an in-vivo parameter

30. Mild mutations are located on the molecule surface30
- Mutations D83N, D175N, S102L, R252T, D313Y, T385A, F396Y,
E398A display only mild effects on GLA activity.
- H-bond disturbances like in the shown mutations are predicted to display only mild effects on enzyme performance
S126G
I219T
R220Q
Provided by Prof. A. Markoff, University of Muenster
Lukas et al., 2015

31. D313Y (Score 5) mutation and Gb3 - Fabry or „Gb3emia“ ?
PATIENT
exon mutation
sex
ALPHAGALACT
Gb3 blood µg/ml
Gb3 IHC skin
Gb3 EM skin
total Gb3
Gb3_24
P0037
c937 G>T [D313Y] m
10,2
3,1
91,32
21,18
P0431 f 27
3,9
negative
P0558 21
3,0
120,00
24,40
P0607 14
2,2
P0691
3,6
6,4
77,00
16,10
P0649 22
2,7
269,11
58,17
P0839
12,7
5,1
unclear
positive
49,00
11,60
P0865 43
130,89
33,15
P1236 17
P1515 15
5,8
228,77
45,59
P3102 19
0,9
P2143
3,4
4,6
317,19
70,63
P4024
11,8
P2704 36
97,76
19,58
P2871 29
P2918
2,4
7,5
67,00
22,30
P3607 48
371,09
79,60 31

32. Two patients with D313Y – 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 nMol MU/mg protein; normal ); Gb3 blood 5.3 µg/ml (normal < 4.0 µg/ml), lyso-Gb3 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** (nMol MU/mg protein); Gb3 blood 4.9 µg/ml (normal < 4.0 µg/ml), lyso-Gb3 1.7 (normal < 1.1 ng/ml)
- MLPA was normal in both cases

33. Skin: Gb3 immunohistochemistry and electron microscopy
Gb3 immunhohistocehmistry
Fabry ctlr. A156V pat AW - D313Y pat UK - D313Y
Electron micrsocopy
EM: typical inclusions with irregular parallel lamellae, characteristic Zebra bodies (Prof. Jonas, Rostock)

34. Lyso-Gb3 of patients on agalsidase alpha (wo back-switch)
males
females

35. Lyso-Gb3 of patients on agalsidase alpha (wo back-switch)
Males (n= 38, 36, 36, 27 with regard to time points)
Females (n=14, 13, 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)

36. Is time on A. alpha related to lyso-Gb3 values. (only values under A
Is time on A. alpha related to lyso-Gb3 values? (only values under A. alpha)
Males
Females

37. CARDIAC PARAMETERS (agalsidase alfa only)
Switch V1 V2 V3
Filling impairment % (n)
26,3% (15)
26,4% (19)
30,4% (21)
24,2% (8)
Valvular lesions % (n)
28,6% (18)
24,3% (18)
29,4% (20)
15,2% (5)
Late enhancement % (n)
8,1% (5)
3,7% (2)
3,0% (1)
ECG changes % (n)
50% (25)
51,3% (40)
59,4% (41)
43,3% (13)
NYHA classification % (n)
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)
NYHA IV
Hypertension % (n)
34,7% (25)
30,2% (26)
33,8% (27)
22,2% (8)
Pacemaker % (n)
6,5% (5)
11,0% (10)
13,4% (11)
NYHA IV raus?

38. 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

39. GFR creatinine clearance in ml/min*1.73m2
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 33 18

40. 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

41. 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 40 23

42. 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

43. Tip of the iceberg model in Fabry
Thx indication Clin Score AOO lysoGb3
+++ ++ +
(+)
Classical phenotype (10%)
Score 10
Oligo-/monosymptomatic phenotype
Late-onset, monosymptomatic phenotype
Score 1