Supplementary Material
Genetic Screening of Anderson-Fabry Disease in Probands Referred from Multi-specialty Clinics.
Valentina Favalli, Eliana Disabella, Mariadelfina Molinaro, Anna Scarabotto, Alessandra Serio, Maurizia Grasso, Nupoor
Narula, Carmela Giorgianni, Clelia Caspani, Monica Concardi, Manuela Agozzino, Calogero Giordano, Alexandra
Smirnova, Takahide Kodama, Lorenzo Giuliani, Elena Antoniazzi, Riccardo Borroni, Camilla Vassallo, Filippo Mangione,
Laura Scelsi, Stefano Ghio, Carlo Pellegrini, Marialuisa Zedde, Laura Fancellu, GianPietro Sechi, Antonello Ganau,
Stefania Piga, Annarita Colucci, Daniela Concolino, Maria Teresa Di Mascio, Danilo Toni, Marina Diomedi, Claudio
Rapezzi, Elena Biagini, Massimiliano Marini, Maurizia Rasura, Maurizio Melis, Antonia Nucera, Donata Guidetti,
Michelangelo Mancuso, Umberto Scoditti, Pamela Cassini, Jagat Narula, Luigi Tavazzi, Eloisa Arbustini
1
METHODS SUPPLEMENT.
GENETIC TESTING
GLA gene
The seven exons of the GLA gene were amplified from peripheral blood-derived genomic deoxyribonucleic acid by means
of polymerase chain reaction. The polymerase chain reaction fragments were analyzed by means of denaturing highperformance liquid chromatography using the wave deoxyribonucleic acid fragment analysis system (Transgenomic, San
Jose, California). Heteroduplex fragments were purified (QIAquick Kit, Qiagen, Santa Clarita, California) and then
sequenced using a BigDye-terminator cycle sequencing system (ABI PRISM, Applied Biosystems, Foster City, California).
Reference sequence used in the study is ENST00000218516.
Definition of mutations and variants
Criteria for defining genetic variants as pathogenic were:

Nonsynonymous genetic variant, both known and previously demonstrated to cause AFD.

Novel proven to be associated with low a-GAL activity and/or intracellular GB3 accumulation.

Truncation-predicting mutation and in-frame deletions predicting loss of key residues for the function of the
protein.

Mutations predicting the substitution of Cysteine (Cys) residues, both involved in disulfide bonds (52-94; 56-63;
202-223; 378-382) and catalytic sites (142-172) as well as unpaired Cys (90 and 174).

Absence of the variant in a large number of controls (1000 Genomes Project, Exome Variant Server (EVS) and
dbSNP137) or minor allele frequency (MAF) <0.01.

Conservation of the affected amino acid in different species through evolution (conservation score);

Predicted pathogenicity (Polyphen 2, SIFT, Mutation Taster).

Pathologic study of affected tissue/s demonstrating intracellular GB3 accumulation using electron microscopy
and immunohistochemistry.

Segregation studies in families demonstrating that mutation carriers show disease manifestations.

Consultation of the GLA mutation database:

http://www.genomed.org/lovd2/variants.php?select_db=GLA&action=view_all

http://fabry-database.org/mutants/
Multigene panels in patients with prominent left ventricular hypertrophy
For the Miseq Illumina we used a custom designed panel with Nextera Rapid Capture Sequencing Kit v2. After library
preparation with Nextera tagmentation method, barcodes were added and sample pulled. Next, libraries were denatured
and biotin-labeled probes specific to the targeted region were used for Hybridization. Biotinylated DNA fragments bound
to the streptavidin-coated beads were magnetically pulled down from the solution. The enriched DNA fragments were
then hybridized and prepared for a second capture. Twelve samples per run were sequenced, according to Illumina
preparation protocols. Sequences were analyzed by mapping all of the generated sequencing reads to the hg19 human
genome reference (UCSC) with dbSNP 138 using GATK(v2.4) for variant calling and Variant Effect Predictor (v85) for
variant annotation and interpretation.
2
The list of genes contained in the custom Sequencing Kit and relative transcriptomes Ensembl reference are: ABCC9
ENST00000261200, ABCG5 ENST00000260645, ABCG8 ENST00000272286, ACTA1 ENST00000366683, ACTC1
ENST00000290378, ACTN2 ENST00000366578, AKAP9 ENST00000356239, ANK2 ENST00000264366, ANKRD1
ENST00000371697, BAG3 ENST00000369085, BRAF ENST00000288602, CACNA1C ENST00000327702, CACNA2D1
ENST00000356253, CACNB2 ENST00000282343, CALM1 ENST00000356978, CALR3 ENST00000269881, CASQ2
ENST00000261448, CAV3 ENST00000343849, CREB3L3 ENST00000078445, CRELD1 ENST00000326434, CRYAB
ENST00000227251, CSRP3 ENST00000265968, CTF1 ENST00000279804, DES ENST00000373960, DMD ENST00000288447,
DNAJC19 ENST00000382564, DOLK ENST00000372586, DPP6 ENST00000332007, DSC2 ENST00000251081, DSG2
ENST00000261590, DSP ENST00000379802, DTNA ENST00000269190, EFEMP2 ENST00000307998, , EMD
ENST00000369835, EYA4 ENST00000355167, FHL1 ENST00000345434, FHL2 ENST00000322142, FKRP ENST00000318584,
FKTN ENST00000223528, FXN ENST00000377270, GAA ENST00000302262, GATAD1 ENST00000287957, GCKR
ENST00000264717, GJA5 ENST00000271348, GLA ENST00000218516, GPD1L ENST00000282541, HCN4
ENST00000261917, HRAS ENST00000311189, HSPB8 ENST00000281938, ILK ENST00000299421, JAG1 ENST00000254958,
JPH2 ENST00000342272, JUP ENST00000310706, KCNA5 ENST00000252321, KCND3 ENST00000302127, KCNE1
ENST00000337385, KCNE2 ENST00000290310, KCNE3 ENST00000310128, KCNH2 ENST00000262186, KCNJ2
ENST00000243457, KCNJ5 ENST00000338350, KCNJ8 ENST00000240662, KCNQ1 ENST00000155840, KLF10
ENST00000285407, KRAS ENST00000256078, LAMA2 ENST00000421865, LAMA4 ENST00000230538, LAMP2
ENST00000200639, LDB3 ENST00000263066, LMNA ENST00000347559, MIB1 ENST00000261537, MURC
ENST00000307584, MYBPC3 ENST00000256993, MYH6 ENST00000356287, MYH7 ENST00000355349, MYL2
ENST00000228841, MYL3 ENST00000292327, MYLK2 ENST00000375985, MYO6 ENST00000369975, MYOZ2
ENST00000307128, MYPN ENST00000354393, NEXN ENST00000294624, NKX2-5 ENST00000329198, NODAL
ENST00000287139, NPPA ENST00000376476, NRAS ENST00000369535, PCSK9 ENST00000302118, PDLIM3
ENST00000284767, PKP2 ENST00000070846, PLN ENST00000357525, PRDM16 ENST00000270722, PRKAG2
ENST00000287878, PRKAR1A ENST00000358598, PTPN11 ENST00000351677, RAF1 ENST00000251849, RBM20
ENST00000369519, RYR1 ENST00000355481, RYR2 ENST00000360064, SALL4 ENST00000217086, SCN1B
ENST00000262631, SCN2B ENST00000278947, SCN3B ENST00000299333, SCN4B ENST00000324727, SCN5A
ENST00000327956, SCO2 ENST00000252785, SDHA ENST00000264932, SEPN1 ENST00000354177, SGCB
ENST00000381431, SGCD ENST00000337851, SGCG ENST00000218867, SHOC2 ENST00000265277, SLC2A10
ENST00000359271, SNTA1 ENST00000217381, SOS1 ENST00000395038, SREBF2 ENST00000361204, TAZ
ENST00000299328, TBX20 ENST00000408931, TBX3 ENST00000257566, TBX5 ENST00000310346, TCAP
ENST00000309889, TGFB3 ENST00000238682, TMEM43 ENST00000306077, TMPO ENST00000261210, TNNC1
ENST00000232975, TNNI3 ENST00000344887, TNNT2 ENST00000236918, TPM1 ENST00000267996, TRDN
ENST00000334268, TRIM63 ENST00000374272, TRPM4 ENST00000252826, TTN ENST00000342175, TTR
ENST00000237014, TXNRD2 ENST00000334363, VCL ENST00000211998, ZBTB17 ENST00000375733. ZHX3
ENST00000309060, ZIC3 ENST00000287538.
PLASMA AND LEUKOCYTE ASSAY
3
The -Gal A activity was determined in plasma and in leukocytes using the synthetic fluorogenic substrate, 4methylumbelliferil--D-galactopyranoside (Sigma-Aldrich, Steinheim, Germany), according to a previously described
protocol with a few changes 1, in the presence of N-acetyl-D-galactosamine (Sigma-Aldrich, Steinheim, Germany), a
specific inhibitor of galactosidase B.
Briefly, for measuring plasma -Gal A, 50 L of plasma were reacted with 300 L of the substrate solution (3 mmol/L in
0.15 mol/L citrate/phosphate buffer, pH 4.8) and 50 L of the inhibitor N-acetyl-galactosamine (0.8 mol/L). The samples
were incubated in bath water at 37 °C for two hours and the reaction was stopped by adding 3.5 mL of 0.2 mol/L
glycine/carbonate buffer, pH 10.7. The leukocytes samples were obtained by the heparinized blood centrifuged at 3000
rpm at room temperature for 10 minutes, and the remaining blood was used to separate leukocytes following a standard
Ficoll-Paque method; the pellet obtained was diluted in distilled water, sonicated, and analyzed for total protein content
trough the colorimetric technique by Lowry et al 2. In the PBMC assay, 25 L of leukocytes homogenate were incubated
for an hour at 37° C with 100 L of substrate solution (7.5 mmol/L in distilled water) and 25 L of Na-acetate buffer,
1mol/L, pH 4.5. Reaction was stopped with the addition of 2 mL of glycine-carbonate buffer, pH 10.7 and the resulted
fluorescence was determined in a Kontron Spectrofluorometer (Uvikon SFM25), (exc: 360 nm; em: 450 nm). Readings
were corrected for blanks and compared with 4-methylumbelliferone calibrator (10 mmol/L). The results of leukocytes
assay represent specific enzyme activity, since they take in consideration the amount of total proteins in a sample, being
expressed as nmol of substrate degraded per hour per mg of the cell protein (nmol/mg prot/h), while the results for
plasma indicate total enzyme activity, being expressed as nmol of substrate per hour per mL of plasma (nmol/mL/h). For
all types of samples, the activity of a reference enzyme, usuallymannosidase, was measured to assure sample quality.
We generated standards for reference values of plasma and leukocytes Gal A activities in wild-type subjects in
accordance with the Standards for Reporting of Diagnostic Accuracy principle.
1.
Desnick RJ, Allen KY, Desnick SJ, Raman MK, Bernlohr RW, Krivit W. Fabry's disease: enzymatic diagnosis of
hemizygotes and heterozygotes. Alpha-galactosidase activities in plasma, serum, urine, and leukocytes. J Lab Clin
Med. 1973;81:157-71.
2.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ, “Protein measurement with the Folin phenol reagent”. J Biol
Chem 1951; 193: 265-75.
PATHOLOGY STUDIES
Tissue biopsies were routinely processed for light and electron microscopy. Light and electron immunochemistry was
performed using anti-Gb3 monoclonal antibody (TCI Chemicals, clone BGR23, formerly sold as Seikagaku cat. #370680)
with DAKOTMEnVision secondary antibody for light microscopy and goat anti-mouse, colloidal gold 15nm (Bb
International) secondary antibody for immuno-electron microscopy.
IN VITRO CELLULAR STUDIES
Blood samples (3 ml) were collected in EDTA-treated tubes and Peripheral Blood Mononuclear Cells (PBMCs) were
isolated by standard density-gradient centrifugation (Histopaque®-1077, Sigma-Aldrich). PBMCs were immediately plated
on cover glass placed in the culture dish with opportune growth medium (Advanced Dulbecco's Modified Eagle
4
Medium ,Gibco, Life Technologies) supplemented with 10% Fetal Bovine Serum, 2mM L-glutamine, 0.1 mg/ml penicillin /
100 U/ml streptomycin and 1x MEM Non-Essential Amino Acids Solution. After 7-10 days of culturing at 37° C in a
humidified 5% CO2 incubator the adherent cells appeared. The cells were fixed and fluorescent staining on this glasses
directly. Nuclei were stained with DAPI solution (Ultracruz Mounting Medium, Santa Cruz).
For the immunofluorescence studies of the Gb3 accumulations, the cells were fixed with 4% paraformaldehyde in PBS 1X
on ice for 15 min and incubated with primary anti-Gb3 antibody (Anti-Gb3 Monoclonal Antibody, clone BGR23, TCI
Chemicals). Anti-mouse Cy3 conjugated (Jackson ImmunoResearch Laboratories) was used as secondary antibody. Nuclei
were stained with DAPI solution (Ultracruz Mounting Medium, Santa Cruz).
For detecting the expression of alpha galactosidase (a-GLA), the cells were fixed with 100% methanol on ice for 10 min
and incubated with primary antibody (Anti-GLA Polyclonal Antibody, Atlas Antibodies). We used anti-rabbit Cy5
conjugated (Jackson ImmunoResearch Laboratories) as secondary fluorescent antibody. All antibodies were diluted in 4%
of Skim Milk Powder (Fluka Analytical) in 1x PBS.
Images were acquired by Nikon D-Eclipse C1 confocal microscopy and EZ-C1 Software.
Statistical analysis
Descriptive statistics were computed as median and IQR for continuous variables. In addition to the main statistical
analysis in the consecutive series of probands (main text), we here used non-parametric statistics for dependent data
(Wilcoxon rank sum test) stratified for family, in case of p.(Asp313Tyr) and p.(Asn215Ser) because they were found in
more families. A p value of <0.05 was considered statistically significant. MEDCALC version 14.10.2 (MedCalc Software,
Belgium) was used for statistical evaluation.
RESULTS SUPPLEMENT.
Organ/system involvement
Of the 37 probands, 12 were the unique family members (including one patient with proven de novo mutation): seven
had family history suggestive for AFD in one non-tested deceased parent; four had no relatives. The 60 mutated relatives
were from 25 families; we traced detailed clinical information for four obligate carriers, which are included in the
evaluation. 5 females accepted genetic testing but refused multidisciplinary clinical controls. Overall, we had clinical
information in 96 mutation carriers: 86 mutated family members were affected including 2 only complaining
acroparesthesias; 10 were healthy carriers. Of the 86 affected family members, 22 (28%) demonstrated the involvement
of one organ (n=14) and 1 organ plus acroparesthesias (n=8), while 62 had involvement of more than one organ (up to 7)
(72%) (Individual data are reported in Table 5 supplement).
GLA activity in patients with [p.(Asp313Tyr)] pseudodeficiency allele
The 8 probands who were carriers of this variant were from the neurology setting. 31 relatives of the 8 probands
underwent genetic screening and enzyme activity testing: overall, 25 family members were carriers and 14 were wild type.
Although numbers are low and non-sufficient for a robust comparative evaluation, we report available data on enzyme
activity, stratified for family members. Plasma for enzyme dosage was available in 10 mutated males from 6 families [2.15
nmol/mL/h (IQR 2-2.7)] and in 7 WT male relatives from 4 families [9.475 nmol/mL/h (IQR 5.46-13.1)] (p<0.001). Plasma
for enzyme dosage was available in 16 mutated females from 7 families [5.55 nmol/mL/h (IQR 2.71-6.7)] and in 6 WT
5
female relatives from 5 families [7.9 nmol/mL/h (IQR 6.475-8.2)] (p=0.033). Leukocytes for enzyme dosage were available
in 8 mutated males from 4 families [17.9 nmol/mgprot/h (IQR 10.525-19.0875)] and in 6 WT male relatives from 3 families
[26.12 nmol/mgprot/h (IQR 23.46-26.76)] (p=0.56). Leukocytes for enzyme dosage were available in 11 mutated females
from 6 families [20.075 nmol/mgprot/h (IQR 15.41-19.35)] and in 5 WT female relatives from 4 families [19.375
nmol/mgprot/h (IQR 11.575-27.2875)] (p=0.92).
GLA activity in patients with [p.(Asn215Ser)]
The 5 probands who were carriers of this mutation came from the cardiology setting. 19 relatives of the 5 probands were
mutation carriers. Although numbers are low and non-sufficient for a robust comparative evaluation, we report available
data on enzyme activity in probands, stratified for family members. Plasma for enzyme dosage was available in 9 mutated
males in 3 families [0.67 nmol/mL/h (IQR 0.42-0.7)] and in 10 mutated females from 3 families [3.32 nmol/mL/h (IQR 34.91)]. Leukocytes for enzyme dosage were available in 7 mutated males from 3 families [3.31 nmol/mgprot/h (IQR 2.874.25)] and in 10 mutated females from 3 families [10.73 nmol/mgprot/h (IQR 8.759-18.32)].
6
TABLE 1 SUPPLEMENT
GLA mutations identified in the project. The majority of the mutations are known and proven as disease-causing mutations. Novel mutations are in bold.
a-Gal Activity
nmol/mL/h (in male
pts)* (F = family; Pt =
patient)
N
Predicted
protein change
c.DNA
Exome Server DB
PolyPhen2
(Class:Score)°
SIFT^
1
p.(Trp44X )
G>A (c.131G>A)
1
p.(Gln57Arg)†
A>G (c.170A>G)
0,51
2,1
no
/
/
no
BENIGN: 0.019
PROBABLY
DAMAGING: 0.999
PROBABLY
DAMAGING: 0.999
PROBABLY
DAMAGING: 1.000
PROBABLY
DAMAGING: 1.000
TOLERATED
1
p.(Cys63Gly)
T>G (c.187T>G)
1,59
no
1
p.(Cys63Tyr)
G>A (c.188G>A)
0,53
no
1
p.(Arg112His)
G>A (c.335G>A)
0,32
TT=0/TC=1/CC=4
059/C=2443
1
p.(Phe113Leu)
T>C (c.337 T>C)
0,6
no
1
p.(Trp162X)
G>A (c.485G>A)
2,9
no
1
p.(Cys172Tyr)
G>A (c.515G>A)
4,16 (female)
1
IVS3+1G>A
G>A (c.547+1G>A)
1
p.(Tyr184Asp)
1
p.(Pro205Leu
fs*35)
Segregation in
this study
http://fabrydatabase.org/mut
ants/
Described by §
Unique family
member
yes
Unique family
member
Unique family
member
Novel
Dysrupting a
disulfide bond
Dysrupting a
disulfide bond
DAMAGING
yes
Later-onset
DAMAGING
Unique family
member
Later-onset
Eng et al.Mol Med 1997, 3: 174-82
/
/
yes
Stop
Rosenberg KM et al. Hum Mutat 2000,
15: 207-8
no
PROBABLY
DAMAGING: 1.000
DAMAGING
yes
Classic
Eng et al.Mol Med 1997, 3: 174-82
0,45
no
/
/
yes
Classic
T>G (c.550T>G)
0,36
no
PROBABLY
DAMAGING: 1.000
DAMAGING
yes
Mutation of a
buried residue
Ashton-Prolla P et al. J Investig Med
2000, 48: 227-35
Tuttolomondo et al. Clin
Biochem. 2012;45:1525-30
(c.613delC)
0,47
no
/
/
yes
frameshift
Present manuscript
no
BENIGN: 0.048
DAMAGING
yes
later-onset
Eng CM et al. Am J Hum Genet 1993,
53: 1186-97
TOLERATED
yes
Novel
Present manuscript
DAMAGING
yes
Classic
Eng CM et al. Am J Hum Genet 1993,
53: 1186-97
DAMAGING
Unique family
member
Novel
Present manuscript
DAMAGING
yes
Classic
Eng CM et al. Hum Mol Genet 1994, 3:
1795-9





Table 2 Suppl.
F.7 pt # 28 = 0,6
F.23 pt# 57 = 0,7
F.25 pt # 74 = 0,6
F.35 pt # 91 = 0,59
F.36 Pt # 97 = 0,78
DAMAGING
DAMAGING
classic
Sakuraba H et al Am J Hum Genet
1990, 47: 784-9
Present manuscript
Present manuscript
Schäfer E et al.Hum Mutat 2005, 25:
412
Eng CM et al.Hum Mol Genet 1994, 3:
1795-9
5
p.(Asn215Ser)
A>G (c.644A>G)
1
p.(Tyr222Asp)
T>G (c.664T>G )
0,5
no
1
p.(Arg227Gln)
G>A (c.680G>A)
0,7
no
1
p.(Phe273Ser)
T>C (c.818T>C)
0,27
no
1
p.(Ala288Asp)
C>A (c.863C>A)
0,93
no
1
p.Ala292_Met29
6del
c.875_886delCTCCT
TTATTCA
0,49
no
/
/
yes
Novel; classic
Present manuscript
8
p.(Asp313Tyr)
G>T (c.937G>T)
AA=0/AC=21/A=
10/CC=4039/C=2
433
PROBABLY
DAMAGING: 0.999
DAMAGING
Yes in 5/8; 3
unique family
members
Polymorphism
 Classic: Eng CM et al. Am J Hum
Genet 1993;53:1186-97
 Neurologic variant: Brouns et al.
Table 2 Supp.
 F.5 pt # 20 = 2,2
 F.5 pt # 21 = 1,1
PROBABLY
DAMAGING: 0.995
PROBABLY
DAMAGING: 1.000
PROBABLY
DAMAGING: 0.999
PROBABLY
DAMAGING: 1.000
7








Stroke 2010; 863-868 and Lenders et
al. PLoS One 8:e5556
 SNP: Yusuda et al. Hum Mutat.
2003;22:486-92.
F.5 pt # 23 = 0,95
F.5 pt # 25 = 0,9
F.8 pt # 32: = 2
F.24 pt # 60 = 2,4
F.24 pt # 66 = 0,99
F.28 pt # 78 = 0,7
F.32 pt # 85 = 2,7
F.6,F.27,F.33:
female pts
1
p.(Phe337Ser)
T>C (c.1010T>C )
0,54
no
1
p.(Arg342Gln)
G>A (c.1025G>A)
0,5
no
1
p.(Ala352Asp)
C>A (c.1055C>A)
0,45
no
1
p.(Arg356Gln)
G>A (c.1067G>A)
11 (female)
no
POSSIBLY
DAMAGING: 0.869
PROBABLY
DAMAGING: 1.000
PROBABLY
DAMAGING: 0.999
PROBABLY
DAMAGING: 0.999
POSSIBLY
DAMAGING : 0.948
PROBABLY
DAMAGING: 1.000
DAMAGING
Unique family
member
Novel
DAMAGING
yes
Classic
DAMAGING
TOLERATED
Unique family
member
Unique family
member
Classic
Classic
Present manuscript
Davies et al. Hum Mol
Genet. 1993;2:1051-3.
Morrone et al. J Med Genet
2003;40:e103
Hwu et al. Hum Mutat 2009; 30:1397
Blaydon D et al.Hum Mutat 2001, 18:
459
Unique family
Topaloglu et al. Mol Med 1999, 5:
1
p.(Cys378Tyr)
G>A (c.1133G>A)
1,39
no
TOLERATED
Classic
member
806-811
Shabbeer J et al. Mol Genet Metab
1
p.(Ser401X)
C>A(c.1202C>A)
4**
no
/
/
yes
Classic
2002, 76: 23-30
PROBABLY
Not specified;
Ebrahim et al. J Inherit Metab Dis
1
p.(Thr410Ile)
C>T (c.1229C>T)
2,3
no
DAMAGING
yes
DAMAGING: 1.000
cardiac phenotype
2012;35:325
Exome Server DB (last access 21 04 2016) (http://evs.gs.washington.edu/EVS/); ° http://genetics.bwh.harvard.edu/pph2; ^http://sift.jcvi.org. § First manuscript that described the mutation. The mutation segregates in family
members. The probands was also tested for known hypertrophic cardiomyopathy genes, and resulted negative. The activity of the enzyme resulted minor then the cutoff level and the variant was not present in EVS. †This mutation
was associated with late onset phenotype and segregated with the phenotype in the family. ** this patient demonstrated severe phenotype and GB3 accumulation in the gastric biopsy (Figure4).
1
p.(Ala377Asp)
C>A (c.1130C>A)
2,3 (female)
no
TOLERATED
yes
Classic
8
TABLE 2 SUPPLLEMENT
A) GLA Intron variants/haplotypes and related plasma enzyme activity
SEX
5'UTR-10C>T
M
x
F
x
5'UTR12G>A
5'UTR-30G>A
c.-105A>G
IVS2: c.370-81_37077 del CAGCC
c.640-16A>G
n
x
c.100022C>T
x
IQR1
IQR3
45
Plasma
nmol/mL/h
8.25
6.87
9.77
x
x
64
8.7
6.9
11.85
M
x
x
36
15.1
12.57
18.85
F
x
x
34
11.1
8.7
13.83
M
x
x
59
11.5
8.6
14.3
F
x
x
88
11.2
8.8
12.9
M
x
14
14.6
13.8
15.03
F
x
8
9.2
7.8
11.17
2
14.8
6
10.33
9.75
10.57
10.5
14.15
9.1
13.35
M
x
x
F
x
M
x
4
13.5
F
x
5
11
M
x
x
x
2
9.85
F
x
x
x
10
12.5
M
x
x
3
7.7
F
x
x
9
10.1
M
x
13
27.9
23.8
30.9
F
x
10
18.6
9.85
22.3
M
x
2
-
F
x
1
-
M
x
x
5
8.9
F
x
x
2
13.2
M
x
5
11.5
F
x
1
-
M
x
x
24
9.1
7.2
10.6
F
x
x
63
8.7
6.9
11.85
M
x
1
13.3
x
x
9
M
x
x
1
x
12.4
B) GLA: Unique synonymous, missense and intron variants identified in the series
Gender
M
F
F
M
M
F
F
Variant
5'UTR-124T>A
c.48T>G p.(Leu16Leu)
c.192C>T p.(Ile64Ile)
IVS2 +76_80del5
p.(Glu203Glu)
IVS6 -58_55del4(AACT)
c.376A>G (p.Ser126Gly)
Clinical setting
Neurology
Neurology
Neurology
Pediatrics
Nephrology
Neurology
Neurology/cardiology*
F
c.640-854_640-853delAG
Neurology°
RNA or EMB or other studies
RNA
None
RNA
None
None
RNA
Absence of Gb3 myocardial accumulation and of diseasespecific neurologic markers
Phenotype based diagnosis of MELAS confirmed as
associated with the MtDNA A3243G mutation
Plasma (nmol/mL/h)
/
18.3
5.3
8.7
15.8
/
4.9
Leukocytes (nmol/mgprot/h)
/
45.3
/
13.9
23.8
/
28.4
7.2
13.4
*reported http://www.genomed.org/lovd2/variants.php?select_db=GLA&action=view_all
10
TABLE 3 SUPPLEMENT
Unique variants identified in eight patients with severe HCM in which we sequenced using Illumina MiSeq and Sanger sequencing for confirming the variants and test relatives.
PolyPhen2
Exome
MAF
benign
-
rs140740776
benign
0.7051
p.Val1125Met
rs121909378
possibly-damaging
-
c.3098G>A
p.Arg1033Gln
rs397516003
possibly-damaging
-
chr10:69959174
c.3335C>T
p.Pro1112Leu
rs71534278
probably-damaging
0.2
Benign
No:F25
MYPN
chr10:69934259
c.2410G>A
p.Glu804Arg
rs62620248
probably-damaging
0.0245
Benign
No:F5
NEBL
chr10:21112137
c.1962T>A
p.Asn654Lys
rs4748728
benign
0.8
Benign
No:F1
NEBL
chr10:21158728
c.523G>A
p.Ala175Thr
rs71541562
benign
0.016
Benign
No:F25
NEBL
chr10:21134282
c.1132G>C
p.Asp378His
rs41277370
probably-damaging
0.0610
Benign
No:F5
NEBL
PDLIM3
RYR1
SGCD
chr10:21139389
chr4: 186429612
chr19: 38976655
chr5:155935708
c.1051A>G
c.503C>T
c.5360C>T
c.287G>A
p.Met351Val
p.Ala168Val
p.Pro1787Leu
p.Arg96Gln
rs4025981
rs34934920
rs45559835
benign
benign
benign
probably-damaging
0.0611
1.66
0.0479
Benign
Benign
Benign/Likely benign
No:F5
No:F36
No:F36
No:F5
TNNT2
TTN
chr1:201330429
chr2:179659912
c.740A>G
c.982C>T
p.Lys247Arg
p.Arg328Cys
rs3730238
rs16866538
benign
possibly-damaging
0.0602
0.0881
Benign
Benign
No:F5
No:F5
TTN
chr2:179658175
c.1492G>A
p.Val498Ile
rs72647851
benign
0.0175
Benign/Likely benign
F23: non assessable §
TTN
chr2:179634961
c.8467G>T
p.Val2823Phe
rs33917087
probably-damaging
0.0155
F23: non assessable §
TTN
chr2:179596554
c.17048A>G
p.Tyr5683Cys
rs72648942
benign
0.0072
TTN
chr2:179395554
c. 20468G>A
p.Ser6748Asn
rs66961115
unknown
-
Benign/Likely benign
Benign/Likely
benign/GVUS
Benign/Likely benign
TTN
chr2:179586604
c.22786G>C
p.Asp7596His
rs72648970
probably-damaging
0.0386
Benign/Likely benign
No:F1
TTN
chr2:179575511
c.28313G>A
p.Arg9438Gln
rs72648998
benign
0.057
Benign/Likely benign
*F31 non assessable
TTN
chr2:179569387
c.29812A>T
p.Thr9938Ser
rs72650006
benign
0.0205
Benign
No: F5
TTN
chr2:179498042
c.42958A>G
p.Lys14320Glu
rs6723526
probably-damaging
0.0813
Benign
F23: non assessable
TTN
chr2:179457147
c.59585C>T
p.Pro19862Leu
rs16866406
benign
0.1473
Benign
*F31 non assessable
Gene
Location
HGVS
HGVS
COX15
chr10:101489475
c.107G>A
p.Arg36His
JPH2
chr20: 42744802
c.1513G>A
p.Gly505Ser
MYBPC3
chr11:47354482
c.3373G>A
MYBPC3
chr11: 47355200
MYPN
DBSNP
ClinVar
interpretation
Segregation (Yes/No)
[family (F) N]
No: F36
Conflicting: Benign(2);
Pathogenic(1)
Confliicting:
Pathogenic(2);Uncertain
significance(1)
Conflicting: Likely
pathogenic(1);Uncertain
significance(1)
No: F36
No: F3
No:F3
No: F7
No:F36
11
TTN
chr2:179436020
c.74839C>T
p.Arg24947Cys
rs744426
probably-damaging
0.1442
Benign
*F31 non assessable
TTN
chr2:179435983
c.74876A>G
p.Asn24959Ser
-
probably-damaging
-
-
*F31 non assessable
TTN
chr2:179406191
c.97613G>A
p.Arg32538His
rs3731749
probably-damaging
0.1453
Benign
*F31 non assessable
TTN
chr2:179401742
c.100094G>A
p.Arg33365Gln
rs55742743
probably-damaging
0.021
Benign/Likely benign
*F31 non assessable
TTN
chr2:179397561
c.103781G>A
p.Arg34594His
rs3829747
probably-damaging
0.1442
Benign
*F31 non assessable
TTN
chr2:179395573
c.105769G>A
p.Glu35257Lys
rs56324595
probably-damaging
0.0175
Benign/Likely benign
No: F25^
TTN
chr2:179395555
c.105787G>T
p.Ala35263Ser
rs67254537
benign
0.0143
Benign/Likely benign
No: F25^
TTN
chr2:179395554
c.105788C>T
p.Ala35263Val
rs66961115
benign
0.0143
Benign/Likely benign
No: F1°
* , ^, °: Endomyocardial biopsy documenting severe Gb3 myocyte accumulation. § the proband is the father of two young unaffected daughters: therefore the segregation with the phenotypes
cannot rely on phenotypic traits. Last access for databases was 30/9/2015. DBSNP ver 138 (http://www.ncbi.nlm.nih.gov/SNP/index.html); MAF (Minor Allele Frequency) from Exome Variant Server
(http://evs.gs.washington.edu/EVS/) ; Polyphen2 (http://genetics.bwh.harvard.edu/pph2/); Sift (http://sift.jcvi.org/); ClinVar (http://www.ncbi.nlm.nih.gov/clinvar/).
12
TABLE 4 SUPPLEMENT - PLASMA AND LEUKOCYTE ENZYME ACTIVITY IN PROBANDS
Plasma
nmol/mL/h
Median (IQR)
Leukocytes
nmol/mgprot/h
Median (IQR)
General population, wild type and mutated probands
M&F
All
Wild Type
Mutated
P value
All
Wild Type
Mutated
P value
Male
probands
N=787
11
(8.3-15.5)
N=769
11.35
(8.4-15.73)
N=18
0.67
(0.54-2.7)
p<0.001
N=444
23.5
(17.05-33.7)
N=431
23.8
(17.9-33.8)
N=13
2.96
(1.28-4.15)
p<0.001
N=714
10.6
(7.73-13.52)
N=697
10.6
(7.8-13.6)
N=17
6.7
(2.6-11.64)
N=421
23.5
(16.42 -31.2)
N=409
24
(17.14-34.4)
N=12
11.8
(9.87-15.3)
Female
probands
p=0.005
p<0.001
13
TABLE 5 SUPPLEMENT. MOGE(S)
The table shows the list of probands and relatives diagnosed with AFD, their position in family pedigrees, and the MOGE(S) descriptor for cardiomyopathies (web-app at
http://moges.biomeris.com/moges.html, app for Android downloadable from Google Play):
M Morphofunctional (M) phenotype of the hypertrophic cardiomyopathy (H) and obstructive (Obs) and non-obstructive (N-Obs) specifications. Overlapping or evolving morphofunctional
profile (i.e. D=Dilated), Early (E) hypertrophic cardiomyopathy (Max LV: 11-14mm), short PR (<PR) interval or Atrio-Ventricular block (AVB) at ECG can be added.
O Organs/systems’ involvement (O):
 heart (H);
 Kidney (K) in case of microalbuminuria, albuminuria, proteinuria; Chronic renal Failure (CRF) renal transplantation (KTx), and dialysis can be further specified;
 Central nervous system (N) either stroke, or transient ischemic attack (TIA), or migraine and imaging markers such as white matter lesions (WML);
 Peripheral nervous system (Acroparesthesia);
 Cutaneous (C) system (e.g. typical angiokeratomas);
 Gastro-enteric system (GE);
 Ocular (E) for Eye describes non-iatrogenic cornea verticillata, E(CV);
 Auditory system (A).
G Genetic (G) or unknown/unproven (U) genetic origin of the disease. In AFD the pattern of inheritance is described as X-linked (XL)
E Etiology (E) in AFD corresponds to mutations in the GLA gene; mutation is in red when pathogenic, yellow in case of genetic variant of unknown significance (GVUS). Green SNP with
some potential functional implication; variants additional to those observed in GLA are added after the GLA mutation.
(S) Stage (S) descriptor includes both the AHA-ACC stage A-D and the NYHA function class (I-IV).
Proband Patient
ID
Sex
MOGE(S)
1
1*
F
2
M
3
M
4
M
5
F
6
F
7
M
8
M
9
M
10
F
11
M
MH(N-Obs)+ D(AVB) OH, C, E(CV) GXL EG-GLA[p.Tyr184Asp] SC-IIb
M0(<PR) OK, E(CV) GXL EG-GLA[p.Tyr184Asp] SA-I
MH(N-Obs) OH, K(CRF), E(CV) GXL EG-GLA[p.Tyr184Asp]
MH(N-Obs) OH, N (stroke), K(Dialysis), Acroparesthesias GXL EG-GLA[p.Arg227Gln] SC-II
MH(E) OH, N (TIA), Acroparesthesias GXL EG-GLA[p.Arg227Gln] SB-I
M0 OAcroparesthesias GXL EG-GLA[p.Arg227Gln] SA-I
MH(E) OH, K, Acroparesthesias GXL EG-GLA[p.Arg227Gln] SB-I
MH(E) OH, K(CRF), Acroparesthesias GXL EG-GLA[p.Arg227Gln] SB-I
M0 OAcroparesthesias, E(CV) GXL EG-GLA[p.Arg227Gln] SA-I
MH(N-Obs) OH, K(CRF) GXL EG-GLA[p.Arg227Gln]
MH(N-Obs)(AVB) OH, K GXL EG-GLA[p.Gln57Arg] SC-III
2
3
N. organs
Involved
(a=acroparesthesias)
3
Age: onset
Age
Age: Comorbidities
of
at
death
symptoms diagnosis
45
65
2
21
23
3
45
OC
67
3+a
12
41
45
2+a
10
51
HPN
a
10
18
HPN, juvenile
2+a
9
29
HPN
2+a
10
31
HPN, juvenile
1+a
10
24
2
60
OC
72
HPN, IHD
2
68(HPN)
88
89
HPN
Dolico-ectasia, basilar artery, HPN
14
4
5
12
F
13
F
14
F
15
F
16
F
17
M
18
F
19
F
20
M
21
M
22
F
23
M
24
F
25
M
6
26
F
7
27
F
28
M
29
F
30
F
31
F
32
M
9°
33
F
10
35
M
34
F
36
F
37
M
12
38
F
13
39
F°
14
40
M
8
11
MH(E)(AVB) OH GXL EG-GLA[p.Gln57Arg] SB-I
MU OU GXL EG-GLA[p.Gln57Arg] (S)
MH(E) OH, K GXL EG-GLA[p.Gln57Arg] SB-I
M0 O0 GXL EG-GLA[p.Gln57Arg] SA-I
MH(E) OH, A GXL EG-GLA[p.Gln57Arg] SB-I
MH(N-Obs) OH, K(CRF), Acroparesthesias, C GXL EG-GLA[p.Arg342Gln] +MT-RNR2[G3010A] SC-II
M0 OK, Acroparesthesias GXL EG-GLA[p.Arg342Gln] SA-I
M0 ON (Migraine, with aura, TIA, WML), GE, Acroparesthesias GXL EG-GLA[p.Asp313Tyr] SA-I
MH(E) OH, K, GE, A GXL EG-GLA[p.Asp313Tyr] SC-II
M0 OK, Acroparesthesias A GXL EG-GLA[p.Asp313Tyr] SA-I
M0(<PR) OGE, Acroparesthesias, C GXL EG-GLA[p.Asp313Tyr] SA-I
M0 OGE, C GXL EG-GLA[p.Asp313Tyr] SA-I
M0 ON (migraine, with aura, WML), Acroparesthesias GXL EG-GLA[p.Asp313Tyr] SA-I
M0(<PR) O GE, acroparesthesias GXL EG-GLA[p.Asp313Tyr] SA-I
M0 ON (strokes, WML) GU EG-GLA[p.Asp313Tyr] SA-I
MH(N-Obs)(AVB) OH GXL EG-GLA[p.Asn215Ser] +MT-RNR2[G3010A] SC-II
MH(E) OH GXL EG-GLA[p.Asn215Ser] +MT-RNR2[G3010A] SB-I
MH(E) OH GXL EG-GLA[p.Asn215Ser] +MT-RNR2[G3010A] SB-I
MH(N-Obs) OH, N (stroke, WML), K GXL EG-GLA[p.Asp313Tyr] SC-II
MU OU GXL EG-GLA[p.Asp313Tyr] SA-I
M0 O0 GXL EG-GLA[p.Asp313Tyr] SA-I
MH(E) OH, N (stroke), K, Acroparesthesias, C, E(CV) GU EG-GLA[p.Trp44X] SA-I
MH(N-Obs) OH, K(Dialysis), GE, C, E(CV) GXL EG-GLA[p.Ala377Asp] SC-III
MH(N-Obs) OH, GE, C, E(CV) GXL EG-GLA[p.Ala377Asp] SB-I
M0(<PR) ON (WML), K, Acroparesthesias, E(CV), A GXL EG-GLA[IVS3 +1G>A] SB-I
M0 O0 GXL EG-GLA[IVS3 +1G>A] SA-I
M0 ON (TIA,WML), K, GE, Acroparesthesias, E(CV) GXL-maternal EG-GLA[p.Cys172Tyr] SA-I
MH(E) OH, N (Stroke,TIA, migraine with aura), Acroparesthesias, GE GXL-maternal EG-GLA[p.Arg356Gln] SB-I
MH(N-Obs) OH, GE, C GXL EG-GLA[p.Arg112His] SB-I
1
53
53
HPN
U
U
82
Refusing evaluation
2
63
63
HPN, Tongue Cancer
0
HC
23
2
62
62
3+a
30
49
1+a
23
23
2+a
37
42
PFO, Raynaud’s
4
55
71
HPN, > cholesterol
2+a
27
57
HPN, left nephrectomy
2+a
50
62
Celiac disease, Gastric B-cell
Lymphoma
Mild Mitro-Aortic disease
2
36
36
1+a
6(migraine)
32
1+a
14
14
1
37
38
PFO; new stroke after PFO repair
1
75
78
HPN
1
36
38
1
75
79
3
16(migraine)
52
HPN
U
U
49
Refusing evaluation
0
HC
22
5+a
30
40
5
49
58
4
41
42
4+a
38
39
0
4+a
59
Thrombosis retinal artery
Prenatal (HC) Confirmed,
post-natal
15
21
3+a
42
54
3
2
4
PKU
15
41
F
15
42
M
16
43
F
44
M
45
M
46
M
47
M
48
F
49
F
50
M
51
F
19
52
F
20
53
M
21
54
M
22^
55
F
56
M
57
M
58
F
59
F
60
M
61
F
62
F
63
F
64
F
65
F
66
M
67
F
68
F
69
F
70
F
17
18
23
24
25
MH(N-Obs) OH GXL EG-GLA[p.Arg112His] SB-I
MH(N-Obs) OH, K(Tx), C, E(CV) GXL-maternal EG-GLA[p.Cys378Tyr] SC-II
M0 OE(CV) GXL EG-GLA[p.Ala292_Met296del in frame] SA-I
M0 OK, GE, Acroparesthesias, C, E(CV) GXL EG-GLA[p.Ala292_Met296del in frame] SA-I
MH(N-Obs) OH, K, Acroparesthesias, C, E(CV), A GXL EG-GLA[p.Trp162X] SC-II
MH(N-Obs) OH, K, Acroparesthesias, C, E(CV), A GXL EG-GLA[p.Trp162X] SC-II
MH(N-Obs) OH, K, Acroparesthesias, C, E(CV), A GXL EG-GLA[p.Trp162X] SC-Iib
MH(N-Obs) OH, Acroparesthesias, C, E(CV), A GXL EG-GLA[p.Trp162X] SC-II
M0 OAcroparesthesias, E(CV) GXL EG-GLA[p.Trp162X] SA-I
MH(N-Obs) OH, K, Acroparesthesias, C, E(CV), A GXL EG-GLA[p.Ala288Asp] SC-III
MH(N-Obs) OH, Acroparesthesias, C, E(CV) GXL EG-GLA[p.Ala288Asp] SA-I
M0 ON (stroke) GU EG-GLA[p.Cys63Gly] SA-I
MH(N-Obs)+D OH, N (TIA, WMLs), K, GE, Acroparesthesias, C, A GXL-Maternal EG-GLA[p.Cys63Tyr] SA-I
MH(E) OH, N (WMLs), K, GE, Acroparesthesias, C, E(CV), A G0 EG-GLA-De novo[p.Ala352Asp]+MT-RNR2[G3010A] SA-I
MH(N-Obs)(AVB) OH, K(CRF), N (WMLs), C, E(CV) GXL EG-GLA-[p.P205LfsX35] SB-I
MH(N-Obs) OH, Acroparesthesias, C, E(CV) GXL EG-GLA-[p.P205LfsX35] SB-I
MH(N-Obs) OH, N (stroke), K, C GXL EG-GLA-[p.Asn215Ser] SC-II
M0 O0 GXL EG-GLA-[p.Asn215Ser] SA-I
M0 O0 GXL EG-GLA-[p.Asn215Ser] SA-I
MH(N-Obs)(AVB) OH, N (stroke), K GXL EG-GLA-[p.Asp313Tyr] SB-I
MU OU GXL EG-GLA-[p.Asp313Tyr] SA-I
MH(N-Obs) OH, GE, Acroparesthesias GXL EG-GLA-[p.Asp313Tyr] SB-I
MU OU GXL EG-GLA-[p.Asp313Tyr] SA-I
MH(N-Obs) OH GXL EG-GLA-[p.Asp313Tyr] SB-I
MU OU GXL EG-GLA-[p.Asp313Tyr] SA-I
M0 O0 GXL EG-GLA-[p.Asp313Tyr] SA-I
MH(N-Obs) OH, C GXL EG-GLA-[p.Asn215Ser] SC-II
MH(N-Obs) OH GXL EG-GLA-[p.Asn215Ser] SB-I
MH(N-Obs) OH, C, E(CV) GXL EG-GLA-[p.Asn215Ser] SC-II
MH(N-Obs) OH, Acroparesthesias GXL EG-GLA-[p.Asn215Ser] SB-I
1
29
29
4
25
35
1
30
31
4+a
2
6
5+a
28
39
5+a
30
41
5+a
32
43
4+a
60
70
1+a
2
2
5+a
40
57
3+a
5
5
1
71
72
6+a
10
46
7+a
11
15
5
12
56
3+a
10
26
Dolico-ectasia Basilar Artery
4
54
60
HPN, >cholesterol,
0
HC
24
0
HC
35
3
48
50
HPN
U
U
47
Refusing evaluation
2+a
60
67
HPN
U
U
58
Refusing evaluation
1
51
55
HPN
U
U
72
Refusing evaluation
0
HC
24
2
68
70
1
47
47
3
37
68
1+a
60
65
73
HPN
16
71
F
72
F
73
M
74
M
26
75
M
27
76
F
28
77
F
M0 O0 GXL EG-GLA-[p.Asn215Ser] SA-I
M0 O N (WML) GXL EG-GLA-[p.Asn215Ser] SA-I
M0 OGE, Acroparesthesias GXL EG-GLA-[p.Asn215Ser] SA-I
MH(E) OH, GE GXL EG-GLA-[p.Asn215Ser] SB-I
MH(N-Obs) OH, N (stroke), K, GE, Acroparesthesias, C GXL-maternal EG-GLA-[p.Phe337Ser] SB-I
M0 ON (Migraine with aura, WML) GU EG-GLA-[p.Asp313Tyr] SA-I
M0 ON (stroke) GXL EG-GLA-[p.Asp313Tyr] SA-I
78
M
79
F
80
M
81
F
82
F
30
83
M
31
84
M
32
85
M
33
86
F
87
F
88
M
89
F
90
F
91
M
92
M
93
M
94
F
95
F
96
F
97
M
98
M
29
34
35
36
0
HC
42
1
41
41
1+a
7
9
2
15
19
5+a
16
42
1
21
26
1
28
29
M0 ON (stroke), K GXL EG-GLA-[p.Asp313Tyr] SC-II
2
60
64
M0 ON (Stroke, WML), K, GE GXL EG-GLA-[p.Asp313Tyr] SA-I
MH(N-Obs) OH, N (stroke), K(Dialysis), Acroparesthesias, C, E(CV), A GXL EG-GLA-[p.Thr410Ile] SC-II
MH(N-Obs) OH, N (Stroke, WML), K(CRF), GE, Acroparesthesias, C, E(CV), A GXL EG-GLA-[p.Thr410Ile] SB-I
3
32
35
6+a
30
50
7+a
73
4
12(angiokerato
mas)
40
6+a
8
55
2
39
62
2
15(HPN)
65
3+a
53
54
0
HC
52
5+a
40
55
1
49
49
1
22
22
3+a
59
69
3
60
64
3
50
59
2
67
OC
0
HC
33
0
HC
35
2
51
60
2
43
59
MH(E) OH, K, GE, C GXL EG-GLA-[p.Thr410Ile] SA-I
MH(N-Obs)(AVB) OH, N (WML), K, GE, Acroparesthesias, C, A GXL-maternal EG-GLA-[p.Phe273Ser] SC-II
MH(N-Obs)(AVB) OH, GE GU EG-GLA-[p.Phe113Leu] SC-II
MH(N-Obs) OH, N (strokes, WML) GXL-maternal EG-GLA-[p.Asp313Tyr] + MT-RNR2[G3010A] SC-IIb
MH(E) OH, N (stroke, WML), Acroparesthesias, A GXL EG-GLA-[p.Asp313Tyr] +MT-RNR2[G3010A] SA-I
M0 O0 GXL EG-GLA-[p.Asp313Tyr] MT-RNR2[G3010A] SA-I
MH(N-Obs)(AVB) OH, N (stroke), GE, Acroparesthesias, E(CV), A GXL EG-GLA-[p.Tyr222Asp] +MT-RNR2[G3010A] SC-II
M0 OE(CV) GXL EG-GLA-[p.Tyr222Asp] +MT-RNR2[G3010A] SA-I
M0 OE(CV) GXL EG-GLA-[p.Tyr222Asp] +MT-RNR2[G3010A] SA-I
MH(N-Obs)(AVB) OH, K, Acroparesthesias, E(CV) GXL EG-GLA-[p.Asn215Ser] SC-IIb
MH(N-Obs) OH, GE, E(CV) GXL EG-GLA-[p.Asn215Ser] SB-I
MH(E) OH, K(CRF), GE GXL EG-GLA-[p.Asn215Ser] SB-I
MH(N-Obs)(AVB) OH, GE GXL EG-GLA-[p.Asn215Ser] SC-II
M0 O0 GXL EG-GLA-[p.Asn215Ser] SA-I
M0 O0 GXL EG-GLA-[p.Asn215Ser] SA-I
MH(N-Obs) OH, N (TIA, WML) GXL EG-GLA-[p.Asn215Ser] SC-II
MH(N-Obs) OH, K GXL EG-GLA-[p.Asn215Ser] SC-II
HPN
65
51
Acoustic Neurinoma, Aneurysm Left
ICA§
HPN, IHD, vertebro-basilar
dolichoectasia
HPN, RA
HPN
52
HPN, NIDDM, >Cholesterol,
Hypotyroidism
56
HPN
Colon cancer at 44 yrs
78
HPN, >sCPK
17
37
99
F
100
F
101
M
MH(N-Obs)(AVB) OH, K(CRF) GXL EG-GLA-[p.Asn215Ser] SC-IV
M0(<PR) O Acroparesthesias, E(CV) GXL EG-GLA-[p.Ser401X] SA-I
M0(<PR) O GE, Acroparesthesias, E(CV) GXL EG-GLA-[p.Ser401X] SA-I
2
60
OC
1+a
47
47
2+a
12
22
76
HPN
*1 proven affected sister, not included in this list. °4 affected brothers. ^ 2 additional affected sisters of II:3; HC: Healthy Carrier. OC= obligate carrier with available clinical information. Hypertension (HPN). IHD=Ischemic Heart Disease. § Giant
Aneurysm of the left internal carotid artery (Diameter 30mm, partly thrombosed) in the suprasellar segment near the origin of the ophthalmic artery causing compression and displacement of the optic chiasm and left optical nerve in the prechiasmatic
segment. sCPK: serum creatin-phosphokinase PKU = Phenylketonuria. RA: Rheumatoid arthritis. NIDDM: Non-insulin-dependent diabetes mellitus.
18
FIGURE 1 SUPPLEMENT.
Endomyocardial biopsy from a patient carrier of the p. (Phe113Leu): the maximal left ventricular thickening was
28mm. A) The panel shows areas of interstitial fibrosis with typical Fabry bodies in the interstitial spaces and myocytes
that demonstrate extensive myofibriallar lysis and Fabry bodies. B) Immunoelectron micrograph showing anti-Gb3
specific labeling of the osmiophilic lamellar bodies.
19
FIGURE 2 SUPPLEMENT.
The figure shows electron (A,B,C,E) and light microscopy (D) views of morphologic (A-C,E) and anti-GB3
immunohistochemistry (D) features of skin biopsies taken from patients carriers of the p.Asp313Tyr. Osmiophilic
lamellar bodies are rare and scattered as confirmed by the spotty immunoreactive bodies seen in the smooth muscle
cells of skin arteriolar vessels (D). Therefore, skin biopsies are not contributory to the understanding of the potential
role of this variant in the phenotype presented by patients.
20
FIGURE 3 SUPPLEMENT.
The figure shows electron micrographs from punch skin biopsies taken to demonstrate the presence of lamellar
bodies in vessels. The samples have been extensively investigated and did not show typical and significant
accumulations of Gb3. D panel shows osmiophilic extracellular material non-sufficient for a morphologic diagnosis.
21
FIGURE 4 SUPPLEMENT.
Pedigree of one of the families with the p. (Asp313Tyr). All carriers are affected and show more than one trait
recurring in AFD (in blue).
22