Article

Screening of Male Dialysis Patients for Fabry Disease
by Plasma Globotriaosylsphingosine
Hiroki Maruyama,* Takuma Takata,† Yutaka Tsubata,‡ Ryushi Tazawa,§ Kiyoe Goto,§ Jun Tohyama,§ Ichiei Narita,|
Hidekatsu Yoshioka,¶ and Satoshi Ishii¶

Summary
Background and objectives Previous reports of Fabry disease screening in dialysis patients indicate that a-galactosidase
A activity alone cannot speciﬁcally and reliably identify appropriate candidates for genetic testing; a marker for secondary screening is required. Elevated plasma globotriaosylsphingosine is reported to be a hallmark of classic Fabry
disease. The purpose of this study was to examine the usefulness of globotriaosylsphingosine as a secondary screening
target for Fabry disease.
Design, setting, participants, & measurements This study screened 1453 patients, comprising 50% of the male
dialysis patients in Niigata Prefecture between July 1, 2010 and July 31, 2011. Screening for Fabry disease was
performed by measuring the plasma a-galactosidase A enzyme activity and the globotriaosylsphingosine
concentration, by high-performance liquid chromatography. Genetic testing and genetic counseling were
provided.
Results A low level of plasma a-galactosidase A activity (#4.0 nmol/h per milliliter) was observed in 47 patients(3.2%). Of these, 3 (0.2%) had detectable globotriaosylsphingosine levels. These patients all had a-galactosidase A
gene mutations: one was p.Y173X and two were the nonpathogenic p.E66Q. The patient with p.Y173X started
enzyme replacement therapy. Subsequent screening of his family identiﬁed the same mutation in his elder sisterand her children. Genetic testing for 33 of the other 44 patients detected 7 patients with p.E66Q. Thus, the plasma
lyso-Gb3 screen identiﬁed Fabry disease with high sensitivity (100%) and speciﬁcity (94.3%).
Conclusions Plasma globotriaosylsphingosine is a promising secondary screening target that was effective forselecting candidates for genetic counseling and testing and for uncovering unrecognized Fabry disease cases.
Clin J Am Soc Nephrol 8: 629–636, 2013. doi: 10.2215/CJN.08780812

Introduction
Fabry disease (FD) is an X-linked lysosomal storage
disorder resulting from a deﬁciency in the activity of
a-galactosidase A (a-Gal A) (1), a lysosomal enzyme. This enzyme deﬁciency causes the systemic
lysosomal accumulation of glycolipids, primarily
globotriaosylceramide (Gb3), in the vascular endothelium and other tissues. Morbidity and mortality
from FD, caused by renal failure, cardiac disease,and early onset stroke, increase with age, andscreens of male patients on dialysis show an FD
prevalence of 0.2%–1.7% (2–11). The advent of aneffective treatment, enzyme replacement therapy(ERT), has increased the importance of identifying
people with FD.
a-Gal A deﬁciency screens have used plasma(2,3,6,7) and dried blood spot (5,9–11) samples; low
leukocyte a-Gal A activity is also used to identify
candidates for further testing (3,5–7,9). Patients positive for the a-Gal A activity screen (i.e., with low activity) are subsequently tested for mutations in thea-Gal A gene (GLA) (2–11). The genetic tests revealed
that a-Gal A activity screening lacks speciﬁcity, inwww.cjasn.org Vol 8 April, 2013that some patients without GLA mutations test positive (5,8,10,11). In addition, many females who areheterozygous for pathogenic GLA mutations havenormal plasma a-Gal A activity, so screening studies
for FD have predominantly focused on male dialysis
patients (3,4,6). A secondary screening method to
identify FD patients is needed. Ideally, this method
would avoid re-sampling or unnecessary genetic testing for FD, which can increase patients’ psychologic
distress.
Although Gb3, found in the plasma of control
participants (12–15), is not a suitable target for secondary screening, globotriaosylsphingosine (lysoGb3) is dramatically increased in the plasma of
male patients with classic FD (12–14), and might
therefore be a good secondary screening target (12).
In this study, we screened the male dialysis population of Niigata Prefecture for FD by measuring theplasma a-Gal A activity and lyso-Gb3 concentrationand veriﬁed the sensitivity and speciﬁcity of the lysoGb3 screen. The study design included methodologic
strategies for reducing patients’ distress.

*Departmentof Clinical
Nephroscience,
Niigata University
Graduate School of
Medical and Dental
Sciences, Niigata,
Niigata, Japan;†
Department ofInternal Medicine,
Nagaoka Chuo
General Hospital,
Nagaoka, Niigata,Japan; ‡Department
of Internal Medicine,
Niigata Prefectural
Central Hospital,
Joetsu, Niigata, Japan;§
Division of Medical
Genetics, Bioscience
Medical ResearchCenter, Niigata
University Medical
and Dental Hospital,
Niigata, Niigata,Japan; |Division ofClinical Nephrology
and Rheumatology,
Niigata University
Graduate School of
Medical and Dental
Sciences, Niigata,
Niigata, Japan; and¶Department ofMatrix Medicine,
Faculty of Medicine,
Oita University, Yufu,
Oita, Japan
Correspondence:
Dr. Hiroki Maruyama,
Department ofClinicalNephroscience,
Niigata University
Graduate School of
Medical and Dental
Sciences, 1-757
Asahimachi-dori,
Chuoku, Niigata,
Niigata 951-8120,
Japan. Email:hirokim@med.niigatau.ac.jp

Copyright © 2013 by the American Society of Nephrology

629

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Clinical Journal of the American Society of Nephrology

Materials and Methods
Study Design
Lyso-Gb3 Screen. Male dialysis patients in Niigata Prefecture, Japan, of unknown FD status, were screened for FD
between July 1, 2010 and July 31, 2011. All 51 dialysis
centers in Niigata Prefecture were invited to participate.
Dialysis patients were informed about the study speciﬁcs,
and those who agreed to participate provided informed
consent. The study design involved four steps: (1) Patientswere screened for FD by measuring their plasma a-Gal
A activity (activity #4 nmol/h per milliliter wasconsidered a positive result); (2) patients with positive results for the a-Gal A activity screen were re-tested by measuring their plasma lyso-Gb3; (3) patients with elevatedlyso-Gb3 were informed in the context of genetic counseling that they might have FD; and (4) with patient consent,
the diagnosis of FD was conﬁrmed by genetic testing of
GLA, and familial diagnosis was performed.
Screen Veriﬁcation. To determine whether the lyso-Gb3
screen missed patients who had FD, we performed genetic
testing on the patients who tested positive in the a-Gal A
activity screen, but negative in the lyso-Gb3 screen.
Plasma Samples
To minimize patient distress, we used plasma obtained
from a single blood sample collection for both the primaryand secondary screening. This procedure reduced thenumber of times that patients had to endure sampling,
permitted the secondary screen to be performed without
alarming the patients unnecessarily, and reduced theburden of labor on the institutional staff.
Determination of Plasma a-Gal A Activity
The a-Gal A activity was assayed with an artiﬁcial substrate, 4-methylumbelliferyl-a-D-galactoside, as described
elsewhere (16). The low cutoff value for a positive plasma
a-Gal A activity was set as the third percentile value (i.e.,
4 nmol/h per milliliter).
Measurement of Plasma Lyso-Gb3
Lyso-Gb3 was extracted from plasma as described (14).
The O-phthaldialdehyde–derivatized lyso-Gb3 was separated by high-performance liquid chromatography (JascoCo., Hachioji, Tokyo, Japan) with a Unison UK-C18
reversed-phase column (15034.6 mm, 3 mm; Imtakt Co.,
Kyoto, Kyoto, Japan), and quantiﬁed by ﬂuorescence detection at lex 340 nm and lem 435 nm, at a 1.0 ml/min ﬂow
rate. The mobile phase was methanol/H2O [88:12 (v/v)].
For quantiﬁcation, authentic lyso-Gb3 (Sigma-Aldrich Co.,
St. Louis, MO) was added to normal plasma.
Genetic Testing
The human GLA consists of seven exons. Exons 1 and 7
were ampliﬁed from genomic DNA by PCR, and a fragment
containing full-length exons 2–6 and all of the exon-intron
boundaries was obtained by RT-PCR using total RNA. The
DNA and RNA were extracted from white blood cells. Genetic testing was performed as described elsewhere (17).
Informed Consent and Ethics
The study protocol was approved by the Ethics Committee of the Niigata University School of Medicine, Oita

University, and the collaborating dialysis centers, andinformed consent was obtained from the patients. Separate
approval and informed consent were obtained for theveriﬁcation protocol. The study protocols were in accordance with the Declaration of Helsinki.
Genetic Counseling before and after Genetic Testing
Patients with detectable plasma lyso-Gb3 were consideredcandidates for genetic testing. The genetic counseling teamconsisted of two certiﬁed specialists in medical genetics, anephrologist, two certiﬁed genetic counselors, two clinicalpsychologists, and a nurse, all from Niigata University. The
principal issues were explained to the examinee, whose consent
and comprehension were conﬁrmed by the team before andafter genetic testing. The genetic testing performed in thisstudy met the recommendations of the Japanese Associationof Medical Sciences (18).
Statistical Analyses
Values are shown as the median and interquartile range(25th to 75th percentiles) or the mean 6 SD. A two-sided
Fisher’s exact test and chi-squared test were used to evaluate the categorical data. P values , 0.05 were consideredsigniﬁcant.

Results
Study Population
Of the 51 Niigata Prefecture dialysis centers, 31 (61%)
participated in this study. Of the total population of male
dialysis patients in Niigata Prefecture (n=2905), 1453 (50%),
including 1428 on hemodialysis and 25 on peritoneal dialysis, were screened. Participation rate among eligible patients in the dialysis centers was 86.7% (1453 of 1676).
Their underlying kidney diseases were chronic GN in
685 patients (47.1%), diabetic nephropathy in 488 patients(33.6%), nephrosclerosis in 142 patients (9.8%), polycystic
kidney disease in 62 patients (4.3%), other disease in 63
patients (4.3%), and unknown in 13 patients (0.9%). Theirmean age at the time of blood sampling was 66.1612.0
years (range, 25.3–95.0 years), and was 57.7615.5 years(range, 12.8–91.4 years) at the start of dialysis. The duration of dialysis was 8.568.4 years (range, 0–42.5 years).
Plasma a-Gal A Activity in the Primary Screening
The median plasma a-Gal A activity of all patients was
7.0 nmol/h per milliliter (5.9–8.4 nmol/h per milliliter). In
47 patients, the activity was #4.0 nmol/h per milliliter,with a median of 3.4 nmol/h per milliliter (2.9–3.9
nmol/h per milliliter). The median plasma a-Gal A activity in the remaining 1406 patients was 7.1 nmol/h per
milliliter (6.0–8.5 nmol/h per milliliter) (Figure 1).
Plasma Lyso-Gb3 Levels in the Secondary Screening
Plasma lyso-Gb3 was detected in 3 of the 47 patients withlow a-Gal A activity (6.4%). Figure 2 shows representative
high-performance liquid chromatography proﬁles of lysoGb3 in plasma samples from the patients in cases 1, 2, and3, the elder sister of the patient in case 1, and a control
participant. The median plasma lyso-Gb3 level in the threepatients was 3.1 nM (1.9–128 nM). Their plasma a-Gal A
activity was lower than that of the patients without

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Screening for Fabry Disease by Lyso-Gb3, Maruyama et al.

631

Figure 1. | Distribution of plasma a-Gal A activities for all patients tested (n=1453) in increments of 1 nmol/h per milliliter, plotted against
the number of patients with activity in each interval (observed range of activity 1.3–88.3 nmol/h per milliliter; median activity 7.0 nmol/h
per milliliter [5.9–8.4 nmol/h per milliliter]). Each interval in the x axis scale refers to the range beginning with the minimum value, but notincluding the minimum value and extending up to the maximum value. For example, 1–2 means the range beginning at 1, but not including 1
and containing the values up to 2. In 47 patients, the activity was #4.0 nmol/h per milliliter, the low cutoff for the test; the median activity inthese 47 patients was 3.4 nmol/h per milliliter (2.9–3.9 nmol/h per milliliter). a-Gal A, a-galactosidase A.

Figure 2. | High-performance liquid chromatography profiles of OPA-derivatized lyso-Gb3 extracted from human plasma. The peakidentified as OPA-derivatized lyso-Gb3 is indicated by the arrow. (A) Case 1 (p.Y173X hemizygote, 128 nM lyso-Gb3); (B) 100 nM authentic
lyso-Gb3 in normal plasma; (C) 50 nM authentic lyso-Gb3 in normal plasma; (D) normal plasma; (E) case 2 (p.E66Q hemizygote, 3.1 nM lysoGb3); (F) case 3 (p.E66Q hemizygote, 1.9 nM lyso-Gb3); (G) case 1’s elder sister (p.Y173X heterozygote, 6.9 nM lyso-Gb3); and (H) 10 nM
authentic lyso-Gb3 in normal plasma. OPA, O-phthaldialdehyde; lyso-Gb3, globotriaosylsphingosine.

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Clinical Journal of the American Society of Nephrology

detectable lyso-Gb3 (n=44), 2.260.8 nmol/h per milliliter(range, 1.3–2.9 nmol/h per milliliter) versus 3.460.6
nmol/h per milliliter (range, 1.9–4.0 nmol/h per milliliter),respectively (P,0.02).

milliliter plasma a-Gal A activity, and 83 nM lyso-Gb3; 2year-old male, 0.0 nmol/h per milliliter plasma a-Gal A
activity, and 140 nM lyso-Gb3.

Case Presentation
A 28-year-old man had suffered from hypohidrosis fromearly childhood. Acroparesthesia was not detected (Table
1). At age 27 years, he suffered from loss of appetite, headache, leg edema, and vertigo. One month later, severe kidney dysfunction, hypertension, anemia, and atrophy of thekidneys were detected at the ﬁrst medical examination.
The diagnosis was stage 5 CKD due to an unknown cause;
he was unfamiliar with FD. He started dialysis immediately.
One month later, he developed restless legs syndrome, which
can be associated with FD (19,20) and insomnia. His plasma
a-Gal A activity was 1.3 nmol/h per milliliter, and lysoGb3 was 128 nM. In his ﬁrst genetic counseling session,
he stated that he hoped the cause of his sickness could bedetermined.
The physical examination disclosed abdominal angiokeratoma and bilateral lower limb lympho-edema. Genetic
testing showed a nonsense mutation in GLA (c.519C.A,
p.Y173X) (21). The Mainz Severity Score Index (MSSI)
score (22) was 32. Thus, his CKD was caused by FD. He
started ERT within 5 months after the primary screening,and his plasma lyso-Gb3 level was 20 nM after 2 months oftreatment. The patient’s parents were divorced, and he
had lived with his father. He was not in contact with hismother, who was suspected to be a heterozygote of the
GLA mutation.
The patient’s elder sister, who did not present any clinical signs of FD, requested genetic counseling and testing.
Her plasma a-Gal A activity and lyso-Gb3 were 4.5 nmol/h
per milliliter and 6.9 nM, respectively. She had the same
GLA mutation as the patient in case 1, as a heterozygote.
All of the sister’s four children had the same GLA mutation,and the three youngest showed only mild heat intolerance
as a sign of FD. The children’s characteristics were as follows: 10-year-old female, 5.2 nmol/h per milliliter plasma
a-Gal A activity, and no detectable lyso-Gb3; 8-year-old
female, 1.3 nmol/h per milliliter plasma a-Gal A activity,and 8 nM lyso-Gb3; 4-year-old male, 0.1 nmol/h per

Verification of Lyso-Gb3 Accuracy
Although this study aimed to avoid excess genetic testing,to determine whether the lyso-Gb3 screen captured all casesof FD in the study population, it was necessary to performgenetic testing on patients who had tested positive in thea-Gal A activity screen, but negative in the lyso-Gb3 screen.
Forty-four of our original patients met this criterion.
The genetic testing was performed between February 1,
2012 and July 31, 2012. Of the original 44 patients, 9
had died, 1 could not provide informed consent due todementia, and 1 did not consent to participate. Thus, we
performed genetic testing for 33 of the original 44 patients.
In 26 patients, there was no mutation in GLA. Seven had amissense mutation in GLA (c.196G.C, p.E66Q) (Table 2)that occurs at an unexpectedly high frequency in Japaneseand Korean patients screened for FD. It is not a pathogenic
mutation, but a functional polymorphism (23,24).
Including the 3 patients who were positive for lyso-Gb3,
we performed genetic testing for a total of 36 male dialysis
patients with low a-Gal A activity. Among them, we found
one patient whose FD had not been diagnosed previously(case 1; see Table 3 for summary). Thus, the plasma lysoGb3 screen identiﬁed FD with high sensitivity (100%) andspeciﬁcity (94.3%).

Discussion

This is the ﬁrst report on the screening of dialysis patientsfor FD by measuring plasma lyso-Gb3. Our ﬁndings showedthat of 1453 male dialysis patients, this method identiﬁed
1 new case of FD, and 2 cases with functional GLA mutation; thus, the prevalence of FD in male dialysis patientswas 0.07%. The patient with FD had a nonsense mutation(p.Y173X), and the other two had a nonpathogenic missense mutation (p.E66Q). All patients with detectable
plasma lyso-Gb3 had a GLA mutation. Finally, 33 patientswith positive screening results for a-Gal A activity but negative results for lyso-Gb3 had a normal GLA gene or carried

Table 1. Characteristics of three patients with a positive result in the lyso-Gb3 screen

Age (yr)
Cause of CKD
Age dialysis started (yr)
Plasma a-Gal A (nmol/h per milliliter)
Plasma lyso-Gb3 (nM)
GLA mutation
Nucleotide change
Amino acid change
MSSI
ERT startedCase 1Case 2

Case 328
Fabry disease
27
1.3
128

49
Focal glomerulosclerosis
30
2.9
3.1

74
Chronic GN
58
2.3
1.9

c.519C.A
p.Y173X
32
Yesc.196G.C
p.E66Q
30
No

c.196G.C
p.E66Q
30
No

The cause of CKD in case 1 was considered unknown before study participation. GLA, a-galactosidase A gene; MSSI, Mainz Severity
Score Index; ERT, enzyme replacement therapy.

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Screening for Fabry Disease by Lyso-Gb3, Maruyama et al.

633

Table 2. Characteristics of the 7 patients with GLA mutation among 33 patients with positive results in the a-Gal A activity but
negative findings in the lyso-Gb3 screen

Patient

1

2

3

4

5

6

7

Age (yr)
Cause of CKD

76
IgA GN80
Chronic
GN

75
Chronic
GN

58
DM

62
Chronic
GN

83
NS

Age dialysis
started (yr)
Plasma a-Gal
A (nmol/h
per milliliter)
GLA mutation
NucleotidechangeAmino acidchangeMSSI
ERT started

52

56

73

76
Bilateral
ureteralcancer
71

53

62

79

2.9

3.4

3.8

2.4

3.8

1.9

2.0

c.196G.C

c.196G.C

c.196G.C

c.196G .C

c.196G.C

c.196G.C

c.196G.C

p.E66Q

p.E66Q

p.E66Q

p.E66Q

p.E66Q

p.E66Q

p.E66Q

18
No

36
No

24
No

24
No

22
No

32
No

26
No

GLA, a-galactosidase A gene; a-Gal A, a-galactosidase A; lyso-Gb3, globotriaosylsphingosine; IgA GN, IgA glomerulonephropathy;
DM, diabetes mellitus; NS, nephrosclerosis; MSSI, Mainz Severity Score Index; ERT, enzyme replacement therapy.

Table 3. Sensitivity and specificity of plasma lyso-Gb3
screening for FD

Lyso-Gb3 (+)
Lyso-Gb3 (2)Total
Sensitivity (%)
Speciﬁcity (%)

FD (+)FD (2)

Total

1
0
1
100

2
33
35

3
33
36

94.3

In total, we performed a-galactosidase A gene analysis for 36
male dialysis patients with low a-galactosidase A activity. We
found one FD (+) patient with class 1 mutation (p.Yl73X) and 35
FD (2) patients consisting of 9 FD (2) patients with class 2
mutation (p.E66Q) and 26 FD (2) patients without a GLA mutation. The sensitivity was calculated by dividing the number oflyso-Gb3 (+) patients by the total number of total FD (+) patients. The speciﬁcity was calculated by dividing the number oflyso-Gb3 (2) patients by the number of total FD (2) patients.
Lyso-Gb3, globotriaosylsphingosine; FD, Fabry disease; GLA,
a-galactosidase A gene.

the functional polymorphism p.E66Q. FD was identiﬁed
exclusively in a patient with detectable plasma lyso-Gb3.
Published reports on male dialysis patients screened for
FD (summarized in Table 4) show that when 0.5% or fewerof the patients were given genetic testing, 100% were positive for FD (2,4,6,7,9). In those in which $4% of the patients were tested, 33% or fewer had diagnosable FD(8,10,11). These results show that a-Gal A activity alone
does not permit the selective identiﬁcation of patients who
are at high risk for FD. Furthermore, because additional
sampling and tests can be upsetting to the patient, westrongly feel that reducing the number of false positives
is important for patients’ peace of mind. In publishedstudies, all but one (3) had a very high rate of re-samplingin cases that were identiﬁed as false positives: 66.7% (6),90.9% (7), 92.5% (5), and 93.1% (9). If the genetic test fromwhich the GLA mutation was not detected was deﬁned as
the unnecessary genetic tests, similarly, unnecessary genetic tests were carried out in a high percentage of patientsin some studies: 60.0% (5), 66.7% (8), 86.7% (11), and 96.0%(10). Unnecessary genetic tests of some studies were 0%(2–4,6,7,9). To avoid false negative results, the screening
cutoff a-Gal A level, the percentage of the control mean orcohort mean was recently set at approximately $50% (7–11). Secondary screening was needed to realize 0 unnecessary genetic tests (7,9). Finally, to educate patients andhelp them manage stress, we strongly feel that genetic
counseling should be given before genetic testing, but
only to patients who are strongly suspected of having or
carrying a mutation for a genetic disease.
In this study, a GLA mutation was found in all of thepatients with detectable plasma lyso-Gb3 levels. Although
our case sample was small, our ﬁndings indicate thatplasma lyso-Gb3 is an effective secondary screening target
for reﬁning and verifying positive results in an a-Gal A
activity screen. In support of the need for a secondary
screen, Andrade et al. (25) reported false negative results
in an a-Gal A activity screen. Secondary screening forplasma lyso-Gb3 levels may solve this problem.
Two classes of GLA mutations are recognized according
to their pathogenicity (26). Class 1 mutations have a highprobability of causing disease. Class 2 mutations are nonpathogenic polymorphisms and include ﬁve missensechanges (p.E66Q, p.S102L, p.S126G, p.D313Y, p.F396Y)(23,27). Therefore, the two patients in this study with detectable lyso-Gb3 levels and the p.E66Q mutation (cases 2
and 3 in Table 1) have not begun ERT.
p.E66Q is a class 2 mutation that retains a high residual
enzyme activity (17). Therefore, plasma lyso-Gb3 could be
difﬁcult to detect in patients with this mutation. However,5.0

Utsumi et al,2000 (2)Nakao et al.,2003 (3)Linthorst et al.,2003 (4)Kotanko et al.,2004 (5)Ichinose et al.,2005 (6)
Tanaka et al.,2005 (7)Bekri et al.,2005 (8)Merta et al.,2007 (9)Fujii et al.,2009 (10)Gaspar et al.,2010 (11)This study

Plasma,2/440 (0.5)
Plasma,6/514 (1.2)
Whole blood,
1/508 (0.2)
DBS,
53/1516 (3.6)
Plasma,3/450 (0.7)
Plasma,11/398 (2.8)
WBC,
3/59 (5.1)
DBS,
58/1521 (3.8)
DBS,
25/625 (4.0)
DBS,
30/543 (5.5)
Plasma,47/1453 (3.2)

a-Gal A

Plasma, WBC,
4/58 (6.9)

WBC,
10/53 (18.9)
WBC,
1/3 (33.3)
WBC,
1/11 (9.1)

WBC,
6/6 (100.0)

a-Gal A

Re-Sampling

Lyso-Gb3,
3/47 (6.4)

Gb3,
6/6 (100.0)

Plasma Gb3,
Lyso-Gb3

3/1453 (0.2)

30/543 (5.5)

25/625 (4.0)

4/1521 (0.3)

3/59 (5.1)

1/398 (0.3)

1/450 (0.2)

10/1516 (0.7)

1/508 (0.2)

6/514 (1.2)

2/440 (0.5)

Positive
beforeGenetic
Testing

3/3 (100.0)

4/30 (13.3)

1/25 (4.0)

4/4 (100.0)

1/3 (33.3)

1/1 (100.0)

1/1 (100.0)

4/10 (40.0)

1/1 (100.0)

6/6 (100.0)

2/2 (100.0)

Positive by
Genetic
Testing

3/1453 (0.2)

4/543 (0.7)

1/625 (0.2)

4/1521 (0.3)

1/59 (1.7)

1/389 (0.3)

1/450 (0.2)

4/1516 (0.3)

1/508 (0.2)

6/514 (1.2)

2/440 (0.5)

New
Discovery
Frequency

54/58 (93.1)

10/11 (90.9)

2/3 (66.7)

49/53 (92.5)

Unnecessary
Re-Sampling (%)

0/3 (0.0)26/30 (86.7)

24/25 (96.0)

0/4 (0.0)

2/3 (66.7)

0/1 (0.0)

0/1 (0.0)

6/10 (60.0)

0/1 (0.0)

0/6 (0.0)

0/2 (0.0)

Unnecessary
Genetic
Testing (%)

Data are presented as sample (% positive), unless otherwise indicated. FD, Fabry disease; a-Gal A, a-galactosidase A; Gb3, globotriaosylceramide; lyso-Gb3, globotriaosylsphingosine; WBC,
white blood cell; DBS, dried blood spot.
aData are presented as the % of control mean or cohort mean.

54.1

48.0

66.0

62.6

55.0

58.2

15.4

35.0

9.6

29.8

Screening
Cutoffa-GalA
LevelaAuthors,
Year

First Sampling

Table 4. Reports on FD screening of male dialysis patients

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Clin J Am Soc Nephrol 8:629–636, April, 2013

we detected plasma lyso-Gb3 in two different families
with the p.E66Q mutation, suggesting that detectable
plasma lyso-Gb3 may directly reﬂect the existence of a
GLA mutation, a point that is very important to verify.
Among our patients testing positive in the a-Gal A activity screen but negative for lyso-Gb3, the only GLA mutation detected was p.E66Q. The fact that the lyso-Gb3
screen missed these individuals may indicate that somepatients with class 2 mutations produce low levels or nolyso-Gb3. Although there is little downside to lyso-Gb3
being undetectable in some patients with class 2 mutations(ERT is not currently indicated for these patients), it isimportant not to overlook class 1 mutations. Further investigations are necessary to clarify whether all class 1
mutations result in the production of lyso-Gb3.
Of seven reports on the FD screening of Japanesemale dialysis patients, including the two most recent(2,3,6,7,10,28,29), the p.E66Q mutation was found in fourstudies (3,10,28,29). Thus, the p.E66Q mutation in this population is frequently detected. If we exclude the FD patientswith p.E66Q in the previous reports, the prevalence of FD
is 0% (10,28,29), 0.2% (6), 0.3% (7), 0.5% (2), and 1.0% (3).
The low prevalence of FD in this study was concerned with
excluding a patient with FD from study population or astudy limitation that not all patients were genotyped, leaving open the possibility that there were some cases of FD
missed.
In the pedigree analysis for the family of the patient incase 1, the children who were hemizygotes for the class 1
FD mutation and the adult heterozygote all had detectable lyso-Gb3 concentrations. Plasma lyso-Gb3 may also
be a promising screening target for adult heterozygotes.
By contrast, lyso-Gb3 was not detected in the 10-year-old
asymptomatic heterozygote, probably because lyso-Gb3
accumulates more slowly in heterozygotes than in hemizygotes (12). Rombach et al. (14) reported that some very
young asymptomatic children who were heterozygous
for a class 1 FD mutation showed normal lyso-Gb3 levels.
Thus, plasma lyso-Gb3 levels vary with the mutation andamong individuals. Therefore, the detection of lyso-Gb3
may become a useful marker for even nonpathologic
GLA mutations, if the detection sensitivity of the test canbe increased.
Plasma lyso-Gb3 is a promising screening tool that was
effective for identifying candidates for genetic counselingand genetic testing, and for uncovering unrecognized FDamong male dialysis patients.
Acknowledgments
We thank Ms. Utako Seino for technical assistance. We are indebted to the patients, nurses, medical engineers, and physicians
who supported this study. The participating centers and physicians
were as follows: Yoshiaki Miura (Miura Medical Clinic), Yutaka
Osawa (Niigata Rinko Hospital), Yutaka Koda (Koda Medical
Clinic), Masaaki Nagai (Aoyagi Clinic), Kensuke Suzuki and
Noriaki Kobayashi (Kitamachi Clinic), Isei Ei (Santo Daini Clinic),
Kentaro Omori and Tsukasa Omori (Omori Clinic), Katsuya
Kimura (Gosen Rokushima Clinic), Takayuki Sato (Nanbugo Kosei
Hospital), Nobuyuki Sakurai (Murakami Memorial Hospital),
Hajime Yamazaki (Nagaoka Red Cross Hospital), Shigemi Kameda(Joetsu General Hospital), Ikuo Aoike (Koyo Medical Clinic), Takashi
Watanabe (Watanabe Medical Clinic), Akira Kamimura and Yoichi

Screening for Fabry Disease by Lyso-Gb3, Maruyama et al.

635

Iwafuchi (Sanjou General Hospital), Syogo Yada (Kido Hospital),
Hirokazu Fujikawa (Maihira Clinic), Kaoru Oya (Kaetsu Hospital),
Masanori Katagiri (Katagiri Clinic), Gen Nakamura and Yoshio
Morioka (Sado General Hospital), Tsukasa Nakamaru (Niigata Prefectural Koide Hospital), Yukihiro Morita (Morita Medical Clinic),
Ryuji Aoyagi (Tachikawa General Hospital), Shin Goto, Noriaki
Iino, and Kazuko Kawamura (Niigata University Medical and
Dental Hospital), Hitoshi Igarashi (Niigata Prefectural Sakamachi
Hospital), Noriyuki Homma (Niigata Prefectural Shibata Hospital),
Taiji Sasagawa (Niigata Prefectural Central Hospital), Yasushi
Suzuki (Saiseikai Niigata Daini Hospital), Asa Ogawa (Keinan
General Hospital), and Michihiro Hosojima and Yuichi Sakamaki(Ojiya General Hospital).
This work was supported by a grant from the Japanese Association of Dialysis Physicians to H.M.

Disclosures
None.
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Received: August 28, 2012 Accepted: November 29, 2012
Published online ahead of print. Publication date available at www.
cjasn.org.