HUMAN MUTATION Mutation in Brief #991 (2007) Online

MUTATION IN BRIEF

Novel Mutations of the GLA Gene in JapanesePatients with Fabry Disease and Their Functional
Characterization by Active Site Specific Chaperone
Masaaki Shimotori1, Hiroki Maruyama2*, Gen Nakamura1, Takayuki Suyama3,
Fumiko Sakamoto3, Masaaki Itoh3, Shigeaki Miyabayashi4, Takahiro Ohnishi5, Norio Sakai6,
Mari Wataya-Kaneda7, Mitsuru Kubota8, Toshiyuki Takahashi9, Tatsuhiko Mori10,
Katsuhiko Tamura11, Shinji Kageyama12, Nobuo Shio12, Teruhiko Maeba13, Hirokazu Yahagi14,
Motoko Tanaka15, Masayo Oka16, Hitoshi Sugiyama17, Toshiyuki Sugawara18, Noriko Mori19,
Hiroko Tsukamoto20, Keiichi Tamagaki21, Shuuji Tanda22, Yuka Suzuki23, Chiya Shinonaga23,
Jun-ichi Miyazaki24, Satoshi Ishii25, and Fumitake Gejyo1
1

Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental
Sciences, Niigata, Japan; 2Department of Clinical Nephroscience, Niigata University Graduate School of Medical
and Dental Sciences, Niigata, Japan; 3Department of Dermatology, Niigata University School of Medicine,
Niigata, Japan; 4Sendai Medical Center, Sendai, Japan; 5Yamada Red Cross Hospital, Tokai, Japan; 6Division ofInternal Medicine, Osaka University Graduate School of Medicine Department of Pediatrics, Suita, Japan;
7
Department of Dermatology Course of Molecular Medicine, Osaka University, Graduate School of Medicine,
Suita, Japan; 8Department of Pediatrics, Hokkaido University School of Medicine, Sapporo, Japan; 9Departmentof Cardiology, Pulmonology, and Nephrology Course of Internal Medicine and Therapeutics Yamagata University
School of Medicine, Yamagata, Japan; 10Third Department of Medicine, Osaka Medical College, Osaka, Japan;
11
Shinonoi General Hospital, Shinonoi, Japan; 12Sio Urological Clinic, Aoi, Japan; 13Asao Kidney Clinic,
Kawasaki, Japan; 14Osaki Citizen Hospital, Cardiovascular Medicine, Osaki, Japan; 15Department of Nephrology,
Akebono Clinic 5-1-1, Shirafuji, Kumamoto, Japan; 16Department of Medicine and Clinical Science, Graduate
School of Medical Sciences, Kyushu University, Fukuoka, Japan; 17Department of Medicine and Clinical Science
Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan;
18
Mutsu General Hospital Department of Cardiology, Mutsu, Japan; 19Kidney Center, Shizuoka General Hospital,
Shizuoka, Japan; 20Depatment of Pediatrics, Sumitomo Hospital, Osaka, Japan; 21Department of Nephrology, St.
Luke’s International Hospital, Tokyo, Japan; 22Omihachiman Community Medical Center, Shiga, Japan;
23
Department of Pediatrics, School of Medicine Ehime University, Ehime, Japan; 24Division of Stem Cell
Regulation Research, Osaka University Medical School G6, Suita, Japan; 25Department of Agricultural and Life
Sciences, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan*Correspondence to: Hiroki Maruyama, MD, PhD, FJSIM. Department of Clinical Nephroscience Niigata
University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Niigata 951-8120, Japan;
Tel.: +81–25–227-0436; Fax; +81-25-227-0437; E-mail: hirokim@med.niigata-u.ac.jp
Communicated by William S. Sly

Fabry disease is an X-linked recessive inborn metabolic disorder caused by a deficiency ofthe lysosomal enzyme α-galactosidase A (EC 3.2.1.22). The causative mutations are diverse,
include both large rearrangements and single-base substitutions, and are dispersed
Received 22 May 2007; accepted revised manuscript 26 September 2007.

© 2007 WILEY-LISS, INC.
DOI: 10.1002/humu.9520

2 Shimotori et al.

throughout the 7 exons of the α-galactosidase A gene (GLA). Mutation hotspots for Fabry
disease do not exist. We examined 62 Fabry patients in Japan and found 24 GLA mutations,
including 11 novel ones. A potential treatment reported for Fabry disease is active site
specific chaperone (ASSC) therapy using 1-deoxygalactonojirimycin (DGJ), an inhibitor of
α-galactosidase A, at subinhibitory concentrations. We transfected COS-7 cells with the 24
mutant GLAs and analyzed the α-galactosidase A activities. We then treated the transfected
COS-7 cells with DGJ and analyzed its effect on the mutant enzyme activities. The activityof 11 missense mutants increased significantly with DGJ. Although ASSC therapy is useful
only for misfolding mutants and therefore not applicable to all cases, it may be useful fortreating many Japanese patients with Fabry disease. © 2007 Wiley-Liss, Inc.
KEY WORDS: α-galactosidase A; GLA; lysosomal storage disease; 1-deoxygalactonojirimycin; chaperone therapy

INTRODUCTION

Fabry disease (FD; MIM# 301500) is a pan-ethnic, X-linked, lysosomal storage disorder caused by a deficiency
in the lysosomal enzyme α-galactosidase A (EC 3.2.1.22) (Brady et al., 1967). Human α-galactosidase A is a
homodimeric glycoprotein with three N-linked oligosaccharide chains on each subunit. Enzyme deficiency results
in a systemic lysosomal accumulation of glycolipids, primarily globotriosylceramide (Gb3), in the vascularendothelium and other tissues. In the classical form of the disease, the patient develops angiokeratoma,
hypohidrosis, and episodic pain crises in the extremities during childhood or adolescence. With advancing age, themorbidity of renal failure, cardiac disease, and early onset of stroke increases. The severity of the clinicalmanifestations depends on the amount of residual α-galactosidase A activity. Hemizygous male patients with noor very low α-galactosidase A activity usually have severe clinical symptoms and die as young adults.
Heterozygous female Fabry patients exhibit a wide range of severity, from a virtually symptom-free course(Marguery et al., 1993) to one comparable to that of their male counterparts (Whybra et al., 2001), although theyusually have no symptoms or very mild manifestations.
We examined Fabry patients in Japan and sequenced the patients’ α-galactosidase A gene (GLA) (MIM#
300644). To analyze the mutant α-galactosidase A activities, we transfected COS-7 cells with the mutant GLAs.
In addition, since 1-deoxygalactonojirimycin (DGJ) stabilizes the α-galactosidase A conformation and improves
its stability (Asano et al., 2000; Ishii et al., 2000; Yam et al., 2006), we added DGJ to the incubation medium andexamined its effect on the mutant α-galactosidase A activities.

MATERIALS AND METHODSPatients

We examined 62 Fabry patients from 31 unrelated families. Diagnosis was based on reduced or absent αgalactosidase A activity and typical signs and symptoms of the disease. We received approval to use the patients’DNA for this study from The Ethics Committee on Genetics of the Niigata University School of Medicine, andobtained informed consent from the patients.
Mutation analysis

Blood samples obtained from patients with Fabry disease were transferred into blood-collecting tubes
containing ethylene diamine tetraacetic acid (EDTA) and stored at 4°C. Unlike heparin, EDTA does not inhibit theactivity of reverse transcriptase, which is used for the reverse transcriptase polymerase chain reaction (RT-PCR).
The human GLA consists of 7 exons. We found it difficult to obtain full-length GLA cDNA (RefSeq
BC_002689.2) by performing a single RT-PCR. We therefore obtained exon 1 and exon 7 by PCR from genomic
DNA, and the region between exons 1 and 7, containing the full-length exons 2-6 by RT-PCR, from total RNA.

Novel Mutations of GLA in Japan

3

White blood cells were collected using Lymphoprep (Axis-Shield PoC, Oslo, Norway). The genomic DNA of
the white blood cells was isolated using an automatic isolation system (NA1000; Kurabo, Osaka, Japan) within 1
week after blood sampling and stored at -20°C until use.
To determine the sequence of exons 1 and 7 of GLA, we performed PCR on genomic DNA with the following
specific primers: Sense Ex1-1(+), 5’-CCAGTTGCCAGAGAAACA-3’; Antisense Ex1-2(-), 5’GAGACTCTCCAGTTCCC-3’; Sense Ex7-5(+), 5’-ACAAGTGCTTGATAGTTCTGA-3’; Antisense Ex7-6(-),
5’-CAGGAAGTAGTAGTTGGCAA-3’. The PCR protocol consisted of 2 min at 94°C, then 30 cycles of 15 sec at
94°C, 30 sec at 59°C, and 30 sec at 68°C with KOD-Plus DNA polymerase (Toyobo, Osaka, Japan). The lengthsof the expected products were 390 bp for exon 1 and 420 bp for exon 7. The PCR products were analyzed by 2%
NuSieve GTG agarose (Takara, Shiga, Japan) gel electrophoresis and then eluted using the Gene Clean Spin Kit(Q-BIOgene, Irvine, CA, U.S.A.).
To determine the sequence of the cDNA region of GLA that included exons 2 to 6, we first amplified it by RTPCR. We generated first-strand cDNA by RT (ReverTra Ace -α-, Toyobo) using random 9-mers. The reaction
consisted of 10 min at 30°C, 20 min at 42°C, 5 min at 99°C, and 5 min at 4°C. To amplify the cDNA of GLA, we
performed the first PCR with specific primers as follows: 5’ #1 primer: 5'-TATGCTGTCCGGTCACC-3', #66(AG66) primer: 5'-TTAAAGTAAGTCTTTTAATGACAT-3'. The PCR protocol consisted of 2 min at 94°C, then
30 cycles of 15 sec at 94°C, 30 sec at 55°C, and 1 min 30 sec at 68°C with KOD-Plus DNA polymerase (Toyobo).
The first PCR products were purified using the QIAquick PCR Purification Kit (Qiagen GmbH, Hilden, Germany).
The length of the expected products was 1.3 kbp. To amplify the GLA cDNA, we performed a second (nested)
PCR with the following specific primers: cDNA#1, 5'-TTGGCAAGGACGCCTAC-3'; cDNA#2, 5'TGCGATGGTATAAGAGCG-3'. The PCR protocol consisted of 2 min at 94°C, then 30 cycles of 15 sec at 94°C,
30 sec at 55°C, and 1 min at 68°C with KOD-Plus DNA polymerase (Toyobo). The length of the expected productwas 1 kbp. The PCR products were analyzed by 2% NuSieve GTG agarose (Takara) gel electrophoresis and theneluted using the Gene Clean Spin Kit (Q-BIO gene).
We analyzed both strands of the GLA sequence in the PCR and RT-PCR products by direct sequencing using an
ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster, CA, U.S.A.).
Construction of pKSCX-GLA

We constructed plasmid pKSCX-GLA by inserting the normal human GLA cDNA in pCXN2Gal (Ishii et al.,1993) into the unique EcoRI site of the pKSCX expression vector, which bears the cytomegalovirus immediateearly enhancer/chicken β-actin hybrid promoter, and the kanamycin-resistance gene for selection (Niwa et al.,1991). We confirmed the pKSCX-GLA construct by sequencing both DNA strands.
Construction of pKSCX-mutant-GLA

To create point mutations or to delete or insert single or multiple amino acids, we used the QuickChange sitedirected mutagenesis kit (Stratagene, La Jolla, CA, U.S.A.) (Yasuda et al., 2004). Because pKSCX contains many
GC-rich sequences, we constructed plasmid pCI-GLA by inserting the normal human GLA cDNA of pCXN2Gal(Ishii et al., 1993) into the unique EcoRI site of the pCI mammalian expression vector (Promega, Madison, WI,
U.S.A.). We then used pCI-GLA as the mutagenesis template. We designed specific primer pairs according toeach mutation. To clone the mutant-GLA cDNA derived from patients with Fabry disease, we incorporated themutation into the pCI-GLA cDNA using the site-directed mutagenesis kit with the specific primer pairs. We
confirmed the mutant GLAs by sequencing both DNA strands. We digested the pCI-mutant-GLA with EcoRI,
eluted the EcoRI-EcoRI fragment containing the mutant-GLA cDNA, and ligated it into the like-digested cloningsite of pKSCX, yielding pKSCX-mutant-GLA.
Cell culture and transfection

We prepared pKSCX-GLA or pKSCX-mutant-GLA using the Qiagen EndoFree plasmid Giga kit (Qiagen
GmbH) (Maruyama et al., 2000), as described previously, and then subjected it to ethanol precipitation.
COS-7 cells (Originator, Gluzman Y; Riken Cell Bank, RCB 0539) were grown at 37°C in Dulbecco’s
modified Eagle’s medium (DMEM; Gibco, Rockville, MD, U.S.A.) containing 10% fetal bovine serum (FBS;
Gibco) (Yasuda et al., 2003). The cells were harvested by trypsin treatment, washed in 10 ml of DMEM
containing 10% FBS, and resuspended at 5×106 cells/ml in DMEM containing 10% FBS. The cells were4 Shimotori et al.

transfected by the LipofectAMINE 2000 method (Invitrogen, Carlsbad, CA, U.S.A.), according to the
manufacturer's instructions. The cells were cultured in DMEM containing 10% FBS at 37°C and 5% CO2. The
COS-7 cells were transfected with pKSCX plasmid in vitro using the LipofectAMINE2000 cationic lipid reagent.
Briefly, 8 µg of DNA (4 µg pKSCX-mutant-GLA and 4 µg pKSCX-luciferase) were mixed with 20 µL of
LipofectAMINE2000 reagent and added to the medium in 60-mm dishes. After the transfection, the transformants
were cultured in 5 ml of DMEM containing 10% FBS with or without 10 µM DGJ (Sigma Chemical, St. Louis,
MO, U.S.A.) at 37°C for 3 days before the enzyme assay. The cells were harvested by trypsin treatment, rinsed
with phosphate-buffered saline, and homogenized with 0.2 ml of water with a Physcotron (NS-310E, Niti-on,
Chiba, Japan). The sample was then subjected to centrifugation at 10000×g for 5 min. The water-soluble extract
was used as the enzyme source.
α-Galactosidase A activity assay

The α-Galactosidase A activity of the water-soluble extract was determined using an artificial substrate, 4methyl-umbelliferyl α-D-galactopyranoside (Nacalai Tesque, Kyoto, Japan), as described previously (Fan et al.,1999). The protein content of the water-soluble extract was measured by the method of Lowry (DC protein assay;
Bio-Rad, Hercules, CA, U.S.A.).
Luciferase assay

Briefly, 20 µl of the water-soluble extract was added to 100 µl of firefly luciferin solution (PicaGene, Toyo
Ink, Tokyo, Japan). The luciferase activity was measured for 10 sec using a luminometer (GENE LIGHT,
Microtec Nition, Chiba, Japan), according to the manufacturer’s instructions.
Statistical analysis

The data are presented as the mean values ± the standard deviation of the mean. We analyzed all data using the
Statcel2 Microsoft Office Excel Add-in Software (Hisae Yanai, Department of Mathematics, Faculty of Science
Saitama University). Statistical significance was evaluated using the unpaired t-test. We considered P values of <0.05 to be statistically significant.
RESULTS AND DISCUSSION

We detected 24 mutations in 62 Fabry patients from 31 unrelated families in Japan (Table 1). The patients’clinical characteristics are shown in Table 2; the numbers correspond to those of the mutant GLAs in Table 1. The
mutations consisted of 17 missense substitutions (p.M42V, p.E66Q, p.M76T, p.D93V, p.R112H, p.S148N,
p.P205T, p.D231V, p.S235F, p.G258V, p.G260A, p.T282A, p.K308N, p.Q312R, p.G328R, p.L403S, p.T410P), 3
nonsense substitutions (p.S102X, p.R227X, p.W399X), 2 small deletions (c.718_719delAA, c.1020delG), 1
insertion (c.323_324insCAGA), and 1 double missense mutation (p.E66Q, p.R112C). Importantly, 11 of themutations (p.M76T, p.D231V, p.S235F, p.G258V, p.T282A, p.K308N, p.Q312R, p.L403S, p.T410P, p.S102X,
c.323_324insCAGA) were novel. The mutant GLA p. E66Q, which is a G-to-C transversion at nucleotide 196(cDNA) of exon 2, was observed in seven putatively unrelated families, and the mutant GLA c.1020Gdel-deletion,
was observed in two putatively unrelated families.
To date, more than 20 mutant GLAs have been reported in Japan (Takata et al., 1997; Okumiya et al., 1995;
Miyamura et al., 1996). Of the 24 mutant GLAs we examined, 4 (p. E66Q, p. G260A, p. G328R, c.
718_719delAA) are the same as the known mutant GLAs in Japan, and 20 are different. Therefore, in the present
study, we examined over half the mutant GLAs in Japan.
To examine the activities of the α-galactosidase A produced by these mutant genes, COS-7 cells weretransfected with the mutant GLAs and cultured with or without 10 µM DGJ for 3 days. All the mutant-αgalactosidase A activities were low, with 0.1% to 50% of the mean normal activity. We examined the relationshipbetween the enzyme activity and clinical symptoms. As expected from previous studies, a one-seventh value of
normal α-galactosidase A activity was sufficient to suppress clinical symptoms, and 8 of the 24 mutations reached
an activity at least one-seventh that of normal α-galactosidase A activity with DGJ treatment (Table 2) (Nakao etal., 1995; Nakao et al., 2003; Yoshitama et al., 2001). The patients with a one-seventh value of normal αgalactosidase A activity or more did not exhibit the classic symptoms of the disease (angiokeratoma, hypohidrosis,

Novel Mutations of GLA in Japan5and episodic pain crises), nor did they develop renal failure and/or cardiac disease later in life (Nakao et al., 1995;
Nakao et al., 2003; Yoshitama et al., 2001).
Some mutant-α-galactosidase A variants form aggregates in the endoplasmic reticulum (ER), due to theirfailure to fold properly during synthesis, and they may lose their enzyme activity. Such mutant-α-galactosidase A
variants may be degraded by the ER quality control system (Yam et al., 2006). Moreover, misfolded αgalactosidase A variants appear to stay within the ER regardless of their catalytic competence, leading to their
possible degradation or aggregate formation. As a result, mutant α-galactosidase A variants with defective folding
tend to be retained in the ER and not reach the lysosomes, where the enzyme functions. DGJ functions as anactive-site-specific chaperone (ASSC) to stabilize the conformation of mutant-α-galactosidase A variants andpreserve at least some of their enzyme activity. In some cases, DGJ allows mutant-α-galactosidase A variants topass through the ER quality control system and be sorted into lysosomes, resulting in an increase in the mutant’s
α-galactosidase A activity. This suggests that the stability of mutant α-galactosidase A is a critical factor that
determines its further transportation and function as residual enzyme activity. However, DGJ does not have ameasurable effect on all mutant α-galactosidase A activities, particularly those with frame-shift or earlytermination mutations.
In this study, the activities of 11 of the 24 α-galactosidase A mutants significantly increased after incubation
with 10 µM DGJ for 3 days (Table 1). The activity of 11 missense mutants significantly increased with DGJtreatment, while that of the other missense, and the nonsense, insertion, and deletion mutants did not increase.
Because the No. 7 mutation causes the incorrect synthesis of most of the protein, including the active site, theapparent enzyme activity of No. 7, 4% of the normal enzyme activity, may be experimental noise. Therefore,
some of the other mutations with less than 4% enzyme activity need to be interpreted carefully. Nevertheless,these findings indicate that although ASSC therapy is not applicable to all patients with Fabry disease, many
Japanese patients might respond to such treatment. Importantly, a preliminary study of DGJ use in transgenic mice
showed no general signs of toxicity (Fan et al., 1999), supporting this compound’s promise as a treatment forhuman Fabry disease.
Our findings support the potential usage of ASSC as an alternative treatment for Fabry disease. It is likely thatby correcting the trafficking defect with DGJ, the misfolded α-galactosidase A variants are directed to lysosomes,
thereby increasing their residual enzymatic activities. Our results showed that DGJ specifically affects misfolded
α-galactosidase A, since it reproducibly increased the activity of different GLA variants. We also showed that
DGJ causes normal α-galactosidase A activity to increase, although the reason is not known. Therefore, DGJ may
be a useful treatment for every heterozygous patient.
Recently, enzyme replacement therapy (ERT) was approved for patients with Fabry disease: two recombinantglycoprotein products, Replagal® (Transkaryotic Therapies, Cambridge, MA, U.S.A.) and Fabrazyme®
(Genzyme, Cambridge, MA, U.S.A.), are now available (Goi et al., 2005; Hoffmann et al., 2006). Because kidneyand heart involvements are important causes of the morbidity and mortality in patients with Fabry disease, theyshould be the main targeted organs in therapies designed to treat it. However, many investigations have reported
that in Fabry disease the kidney and heart are highly resistant to ERT with either enzyme treatment (Brady et al.,
2001; Ioannou et al., 2001). Furthermore, most of the injected enzyme is delivered to the liver and has difficulty
reaching other organs, such as the kidney and heart (Lee et al., 2003; Sakuraba et al., 2006). In addition, the Gb3
in the Fabry mouse kidney is highly resistant to ERT by systemic intravenous injection. In ERT of the Fabry
mouse, most (65~70%) of the injected enzyme is recovered in the liver, and less than roughly 1% of the injected
dose is detected in the kidney and heart (Lee et al., 2003). An examination of ERT in the Fabry mouse also
showed that only a very small quantity of the recombinant enzyme remains in tissues hours after the infusion.
DGJ treatment, in contrast, increased the enzyme activity of normal α-galactosidase A and of several mutant-αgalactosidase A variants (Table1, No.1, 4, 8, 10, 13, 15, 16, 17,18, 19, 23) enough to extend the persistence of
enzyme activity in vitro. Using DGJ as an ASSC may resolve the problems caused by mutant α-galactosidase A
effectively. Increasing the activity of both normal and mutant α-galactosidase A with DGJ treatment may prove
clinically useful.

6 Shimotori et al.

ACKNOWLEDGMENTS

This work was partly supported by a Grant-in-aid for Scientific Research (C) from the Ministry of Education,
Science, Sports, and Culture to Hiroki Maruyama.

Table 1. Mutations in the GLA genes of Fabry patientsNo.

Number ofPatientsExon(hemizygote/heterozygote)

Nucleotide
Change(RefSeq
BC_002689.2)

Amino acidchange(RefSeq
AAH02689.1)

GLA activity
DGJ (-)DGJ (+)
7

nmol/h/ml/10 RLU
normal
GLA
1
2
3

1/0
8/8
1/0

4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24

0/1
1/0
0/1
1/0
1/0
2/1
0/1
1/0
1/0
1/0
3/0
1/1
1/0
1/1
1/0
2/0
1/0
1/2
1/3
2/0
6/5

1
2
2
2
2
2
2
2
2
3
4
5
5
5
5
5
5
6
6
6
6
7
7
7
7

c.124A<Gc.196G<C
c.196G<C
c.334C<Tc.227T<C
c.278A<Tc.305C<Gc.323_324insCAGA
c.335G<A
c.443G<A
c.613C<A
c.679C<Tc.692A<Tc.704C<Tc.718_719delAA
c.773G<T
c.779G<C
c.844A<G
c.924A<Tc.935A<Gc.982G<A
c.1021delG
c.1196G<A
c.1208T<C
c.1228A<C

p.M42V
p.E66Q
p.E66Q
p.R112C
p.M76T
p.D93V
p.S102X
p.R112H
p.S148N
p.P205T
p.R227X
p.D231V
p.S235F
p.G258V
p.G260A
p.T282A
p.K308N
p.Q312R
p.G328R
p.W399X
p.L403S
p.T410P

GLA DGJ (-) GLA DGJ (+) GLA DGJ (+)/normal GLA /normal GLA /GLA DGJ (-)
%

%

ratio

181.0

1.8

3770 ± 1410

*6840 ± 1980

160 ± 52
1883 ± 102
121 ± 53*848 ± 245
2280 ± 841
129 ± 30

4.2
49.8
3.2

22.5
60.5
3.4

5.3
1.2
1.1

108 ± 49
45 ± 771 ± 2789 ± 60
24 ± 35±6
321 ± 10881 ± 33
128 ± 32120 ± 23
19 ± 1173 ± 12406 ± 659±2
175 ± 38381 ± 57
8 ± 1227 ± 1076 ± 31857 ± 262
6±3*222 ± 31
56 ± 774 ± 24150 ± 119*2470 ± 716
49 ± 7*5640 ± 776
104 ± 29160 ± 33*305 ± 63
20 ± 13***385 ± 24***2230 ± 257*223 ± 62***1770 ± 125***1640 ± 197
9±6
78 ± 1186 ± 28*1440 ± 176
8±3

2.9
1.2
1.9
2.4
0.6
0.1
8.5
2.1
3.4
3.2
0.5
1.9
10.8
0.2
4.6
10.1
0.2
0.7
2.0
22.7
0.2

5.9
1.5
2.0
4.0
65.5
1.3
150.0
2.8
4.2
8.1
0.5
10.2
59.2
5.9
46.9
43.5
0.2
2.1
2.3
38.2
0.2

2.1
1.2
1.1
1.7
102.0
9.1
17.6
1.3
1.3
2.5
1.0
5.3
5.5
25.3
10.1
4.3
1.1
2.9
1.1
1.7
1.3

*, p < 0.05 **, p < 0.005 ***, p < 0.0005.
The α-galactosidase A activity of COS-7 origin is subtracted from the results of the α-galactosidase A activity assay.

Novel Mutations of GLA in Japan

7

Table 2. Clinical characteristics of the study patients
serum
GLA
activityNo*

age atonset

neuropathicpainhypohidrosis

Angiokeratoma

cornealopacityurine
abnormality

ECG(nmol/h/mg)
1

hemizygote

unknown

10

-

-

+

+

protein2+,occult
blood1+

left ventricular
hypertrophy

2

hemizygote

3.4

26

-

-

-

-

-

normalhemizygote

2.3

45

-

-

-

-

-

left ventricular
hypertrophy

hemizygote

1

-

-

-

-

-

-

normalhemizygote

3.5

-

-

-

-

-

-

normalhemizygote

unknown

-

-

-

-

-

-

normalleft ventricular
hypertrophy

hemizygote

unknown

50

-

-

-

-

chronic renal
failure on HD

heterozygote

unknown

-

-

-

-

-

-

normalheterozygote

unknown

-

-

-

-

-

-

normalheterozygote

unknown

-

-

-

-

-

-

unknownheterozygote

unknown

50

-

-

-

-

chronic renal
failure on HD-

heterozygote

2.5

44

-

-

-

-

-

unknownheterozygote

4.3

47

-

-

+

-

unknown

unknownheterozygote

4.5

21

-

-

-

-

unknown

unknownheterozygote

4

24

-

-

-

-

unknown

unknown

chronic renal
failure on HD
chronic renal
failureleft ventricular
hypertrophy
left ventricular
hypertrophy
left ventricular
hypertrophy

heterozygote

unknown

40

-

-

-

-

heterozygote

4

40

-

-

-

-

3

hemizygote

unknown

8

+

+

-

-

protein2+

4

hemizygote

unknown

20

-

-

-

-

protein2+

normal5

heterozygote

11.1

unknown

-

-

-

-

unknownnormal6

heterozygote

8.4

11

+

-

-

+

-

normal7

hemizygote

0.2

9

+

-

+

+

protein2+

8

hemizygote

unknown

50

-

-

-

-

chronic renal
failure on HD

left ventricular
hypertrophy
left ventricular
hypertrophy

9

hemizygote

0.8

childhood

+

-

+

+

-

normalhemizygote

1.6

childhood+-+

unknown

protein2+

normalheterozygote

11.4

+

-

+

-

left ventricular
hypertrophy

8 Shimotori et al.

serum
GLA
activityNo*

age atonset

neuropathicpainhypohidrosis

Angiokeratoma

cornealopacityurine
abnormality

ECG(nmol/h/mg)
left ventricular
hypertrophy
left ventricular
hypertrophy
aorticregurgitation 3

10heterozygote

unknown

40

unknown-

unknown

unknown-11

hemizygote

unknown

11

+

+

-

-

protein3+,occult
blood+-

12

hemizygote

3.7

3

+

+

+

+

protein+-13

hemizygote

unknown

14

+

+

+

-

unknownnormal14

hemizygote

2

9

+

unknown---normalhemizygote

2.2

10

+

unknown-+-normalhemizygote

2.5

13

+

unknown---normalheterozygote

4.3

16

-

-

-

+

-

normalhemizygote

3.4

+

+

+

-

protein2+

16

hemizygote

1.2

7

+

+

-

+

protein+,occult
blood+

17

heterozygote

5.25

45

-

-

-

-

-

hemizygote

3

12

+

+

-

-

-

18

hemizygote

unknownchildhood+-

unknown

unknown

protein2+

19

hemizygote

0.4

-

-

-

-

-

-

-

hemizygote

1.8

-

-

-

-

-

-

-

20

hemizygote

5

unknown

-

-

-

-

-

normal21

hemizygote

0.4

25

-

-

-

-

protein2+

heterozygote

6.3

10

+

-

-

+

protein3+

heterozygote

11.7

10

+

-

-

+

protein3+

hemizygote

0.3

8

+

+

-

-

-

-

heterozygote

unknown

unknown

-

-

-

-

unknown

unknownheterozygote

unknown

unknown

-

-

-

-

unknown

unknownheterozygote

unknown

unknown

-

-

-

-

unknown

unknown

hemizygote

3.1

unknown

-

-

-

-

protein+hemizygote

unknown

unknown

-

-

-

-

-

hemizygote

2.4

10

+

+

+

-

protein3+

heterozygote

unknown

8

+

+

+

+

chronic renal
failure on HD

15

22

23

24

left ventricular
hypertrophy
left ventricular
hypertrophy
mitralregurgitation 3
left ventricular
hypertrophy
left ventricular
hypertrophy

left ventricular
hypertrophy
left ventricular
hypertrophy
left ventricular
hypertrophy

left ventricular
hypertrophy
left ventricular
hypertrophy
sick sinus
syndrome
left ventricular
hypertrophy

Novel Mutations of GLA in Japan

serum
GLA
activityNo*

age atonset

neuropathicpainhypohidrosis

Angiokeratoma

cornealopacityurine
abnormality

chronic renal
failure on HD
chronic renal
failure on HD

9

ECG(nmol/h/mg)
heterozygote
heterozygote

5
3.1

20
10

+
+

+

+
+

+
+

hemizygote

2.9

childhood

+

+

+

+

hemizygote
heterozygote
heterozygote
hemizygote
heterozygote

2
3.8
3
3
4.2

13
8
12
7
12

+
+
+
+
+

+
+
+
-

+
-

-

hemizygote

unknown

12

+

+

+

+

chronic heart
failure-*The mutation numbers in Table 2 correspond to those in Table 1.
Case numbers in bold italic denote α-galactosidase A activity greater than one-seventh of normal, following DGJ treatment.

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