REVIEW ARTICLE WCN 2013 Satellite Symposium ‘‘Kidney and Lipids’’

Fabry disease: experience of screening dialysis patients for Fabrydisease

Eiji Kusano • Osamu Saito • Tetsu Akimoto •

Yasushi Asano

Received: 25 September 2013 / Accepted: 18 October 2013

� Japanese Society of Nephrology 2013

Abstract The prevalence rate for Fabry disease is con-

ventionally considered to be 1 case in 40,000; however, due

to increased screening accuracy, reports now suggest that

prevalence is 1 case in 1,500 among male children, and it is

likely that the clinical importance of the condition will

increase in the future. In dialysis patients to date, prevalence

rates are between 0.16 and 1.2 %. Globotriaosylsphingosine

(Lyso-GL-3), which is a substrate of a-galactosidase A (a-

Gal A), has surfaced as a new biomarker, and is also effective

in the determination and monitoring of the effects of enzyme

replacement therapy. In terms of genetic abnormalities, the

E66Q mutation has recently become a topic of discussion,

and although doubts have been expressed over whether or not

it is the gene responsible for Fabry disease, there is still a

strong possibility that it is a functional genetic polymor-

phism. At present, the standard treatment for Fabry disease is

enzyme replacement therapy, and in order to overcome the

problems involved with this, a method of producing

recombinant human a-Gal A using methanol-assimilating

yeast, and chemical or medicinal chaperone treatment are of

current interest. Migalastat hydrochloride is known as a

pharmacological chaperone, but is currently in Phase III

global clinical trials. Adding saposin B to modified a-N-

acetyl galactosaminidase is also under consideration as a

treatment method.

Keywords Fabry disease � Dialysis patients �Screening � a-Galactosidase A � Lyso-GL-3 �E66Q mutation � Enzyme replacement therapy �Chemical chaperone treatment

Introduction

Fabry disease is a form of lysosomal disease, in which the

activity of hydrolase a-galactosidase A (a-Gal A), which

exists within lysosomes, either falls or is deficit, with the

result that the glycolipid conversion of globotriosylcera-

mide (GL-3) to lactosylceramide (GL-2) is impeded. GL-3

then accumulates in various organs and cells throughout

the body, giving rise to a range of disorders over a period

of several decades. The condition is an X-linked recessive,

extremely rare condition, but is frequently the cause of

stroke, renal failure and heart attack in young people. Since

the condition has a major negative impact on a patient’s

quality of life, early detection and treatment are vital. Since

2004, a-Gal enzyme replacement therapy has been offered

under Japanese health insurance, offering the possibility of

early detection and thereby avoiding the dangers of death

from renal failure, heart attacks and strokes.

Fabry disease subtype

The condition known as Fabry disease was first reported by

the British and German dermatologists, Anderson and

E. Kusano (&)

Utsunomiya Social Insurance Hospital, 11-17, Minami

Takasago, Utsunomiya, Tochigi 321-0143, Japan

e-mail: eishun@jichi.ac.jp

E. Kusano

Jichi Medical University, Shimotsuke, Japan

O. Saito � T. Akimoto

Division of Nephrology, Department of Medicine, Jichi Medical

University, Shimotsuke, Japan

Y. Asano

Koga Red Cross Hospital, Jichi Medical University, Shimotsuke,

Japan

123

Clin Exp Nephrol

DOI 10.1007/s10157-013-0897-2

Fabry, in 1898. They noted the characteristic dermatolog-

ical observations that we now associate with Fabry disease,

along with angiokeratoma corporis, in patients.

The genes that encode a-Gal A are present in the X chro-

mosome long arm Xq121.33-q22, meaning that Fabry disease

has an X-linked recessive, and that severe symptoms occur in

male hemizygote patients. Patients with Fabry disease have

deficient a-Gal A activity in white blood cells, blood serum,

urine and organ tissue throughout the body, while carriers

demonstrate intermediate values. This reduction in enzyme

activity results in GL-3 and other glycolipid substrates accu-

mulating in tissue and organs, causing symptoms in the skin,

eyes, digestive organs, heart and kidneys, as well as psycho-

logical and neurological symptoms.

Not all Fabry disease patients demonstrate the same

clinical symptoms, however, and clinical presentation can

be broadly divided into ‘classical’, ‘cardiac-variant’ and

‘renal-variant’. Classical type demonstrates the classical

symptoms of Fabry disease, beginning with pain in the four

limbs during early school years, and progressing to hypo-

hidrosis, angiokeratoma, corneal clouding and other

symptoms. With increasing age, the patient may suffer end-

stage kidney disease (ESKD), ischemic heart disease and

cerebrovascular disorders, and many patients die of these

conditions. With renal-variant, symptoms are not noted

throughout the body; the main attribute of the condition is

ESKD. There are some, although not many, dialysis

patients who have renal Fabry disease in addition to clas-

sical Fabry disease, and there is the possibility that some

may be undergoing dialysis for undiagnosed reasons.

Cardiac variant is similar to renal variant, with symptoms

not noted throughout the body as they are with classical

type, but from middle age onwards patients tend to suffer

mainly from cardiac disorders, most commonly hypertro-

phic cardiomyopathy. The mechanisms by which subtypes

other than classical type occur are extremely interesting; it

has been conjectured to date that mutations without par-

ticular impact on a-Gal A activity, in other words E66Q

and other mutations play a role, although this has not been

clarified [1].

Fabry disease prevalence rate and screening

The prevalence rate for Fabry disease is conventionally

considered to be 1 case in 40,000; however, a report of

recent screening of newborns now suggests the prevalence

to be 1 case in 3,100 [2]. Furthermore, screening of male

children has led to a report that prevalence is 1 case in

1,500 [3]. Improvements in screening accuracy are thought

to be facilitating the diagnosis of this disease. The fre-

quency of the condition is higher than was previously

believed, and in the future the clinical importance of the

condition is likely to rise.

The targets of screening for Fabry disease have been

newborns, patients who have suffered early-onset stroke,

patients undergoing dialysis for ESKD, and patients

developing left ventricular hypertrophy at a young age,

among others (Fig. 1). Ascertaining Fabry disease is easi-

est, in order, among patients suffering early-onset stroke,

patients with unexplained left ventricular hypertrophy,

dialysis patients and newborns (Table 1). While screening

of patients with cardiac hypertrophy and stroke patients

delivers a high rate of positive results, implementing this

on a nationwide level is problematic. The positive rate in

screening newborns is low, and follow-up is difficult. Other

problems include the psychological stress under which this

places parents.

From screening of dialysis patients to date, we have

found that between 0.16 and 1.2 % have Fabry disease, and

there may be more cases of latent Fabry disease (renal

variant) among patients with ESKD, presenting no classical

symptoms, than was previously imagined. According to a

statistical survey carried out by the Japanese Society for

Dialysis Therapy, of approximately 295,000 dialysis

patients in Japan at the end of 2011, only 0.1 % (289 cases)

had congenital metabolic disorders, including Fabry dis-

ease, while at the same time 8.3 % (24,417 cases) were

classified as having an unknown primary disease [4].

Against this background, the authors began to imple-

ment the nationwide epidemiological study into Fabry

disease referred to as Japan Fabry disease screening study

(J-FAST), focusing on dialysis patients. We screened 8,547

dialysis patients (male 5,408, female 3,139), among whom

we found 25 patients (11 male and 14 female, 0.3 %) who

gave a positive result in the third test. Of the 25 patients, 11

did not accept genetic analysis. The remaining 14 patients

agreed to genetic analysis, which revealed that 3 patients (2

male, 1 female had a variation of the a-Gal gene and 7

patients showed E669 variations (4 male, 3 female), and 4

patients (2 male, 3 female) had no variations denying Fabry

disease. The prevalence rate of both male and female

dialysis patients (3 patients) with Fabry disease in this

study was 0.04 % (male 0.04 %, female 0.03 %) other than

those with the E66Q mutation. These results in fact dem-

onstrated a lower prevalence rate for both sexes compared

with other reports to date. A major potential factor in this

was considered to be that screening to date might have

included the E66Q mutation, which has been frequently

studied in Japan. In addition, since 11 of 25 patients did not

receive genetic analysis, there may be some patients with a

variation of the a-Gal gene and E66Q mutation. Therefore,

taking this into account could explain the lower prevalence

rate in J-FAST.

Clin Exp Nephrol

123

Diagnosis of Fabry disease

Generally, a confirmed diagnosis of Fabry disease can be

achieved either by histopathologically demonstrating the

accumulation of substances in the organs, biochemically

demonstrating a reduction in activity in blood a-Gal A or

abnormal elimination of GL-3 in urine, or genetically

demonstrating the genetic mutation of a-Gal A. Diagnosis

is usually performed by measuring a-Gal A activity in

blood serum, and if it is low, measuring white blood cell

activity, and if this is reduced, implementing genetic test-

ing. Alternatively, since the a-Gal A substrate glob-

otriaosylsphingosine (Lyso-GL-3) also accumulates

similarly to GL-3, a high level may indicate Fabry disease.

Lyso-GL-3 is an effective new biomarker for Fabry disease

[5]. Furthermore, it is effective in determining the effec-

tiveness and monitoring of enzyme replacement therapy

[6].

Fabry disease demonstrates an X chromosome genetic

form, with the result that its symptoms are severe in

hemizygote males, but in females, who are heterozygotes,

its symptoms can range from none through to a similar

level of severity to hemizygotes.

The problem of E66Q mutation

Around 600 different genetic abnormalities have so far

been reported, but recently the E66Q mutation, which has

been found more frequently than suspected in Japan and

Korea, is a particular problem. Attention is focused on

whether or not it is a causative gene for Fabry disease,

since a-Gal A activity is lower than among healthy people,

or alternatively, whether it is a functional genetic poly-

morphism. The Sakuraba group suspected that patients

with the E66Q mutation may have either renal or cardiac

variant delayed-onset Fabry disease, and in order to

investigate this they implemented biochemical, pathologi-

cal and structural research. As a result, they demonstrated

that causing the E66Q mutation in a-Gal A enzymes results

in slight changes to the formation of the surface of these

enzymes, and causes instability in the enzyme proteins.

Biochemically, however, a relatively high level of enzyme

activity was maintained in white blood cells, and GL-3 did

not accumulate in a cultured fibroblast. Even under elec-

tron microscopy, no zebra body was noted in skin tissue.

Given these facts, it was concluded that the E66Q mutation

is not a causative gene for Fabry disease, but rather a

functional genetic polymorphism [1, 6, 7].

Treatment

Anti-inflammatory analgesics have almost no effect on the

acroparesthesia in all four limbs at early stages of the

disease, but in many cases regular carbamazepine is

effective. Cardiac symptoms are treated with calcium

antagonists, angiotensin-converting enzyme inhibitors,

angiotensin receptor blockers or beta-blockers. Once

Fig. 1 Onset of signs and

symptoms in typical Fabry

disease

Table 1 Prevalence rates for Fabry disease in newborn infants,

patients on dialysis, patients with left ventricular hypertrophy and

patients with criptogenic stroke

Objects Prevalence rates for Fabry

disease (%)

Newborn infants 0.0025–0.032

Patients on dialysis 0.16–1.2

Patients with left ventricular hypertrophy 1.4–12

Patients with cryptogenic stroke 3.65

Clin Exp Nephrol

123

patients develop ESKD they are treated with dialysis and/

or renal transplantation. Thrombosis is treated with ticlo-

pidine or other antiplatelet drugs.

(a) Gene transfer

At present, gene therapy has been studied using a mouse

model of Fabry disease in which a-Gal A has been knocked

out. However, in experiments using retrovirus, adenovirus

and adeno-associated virus as vectors, in which a-Gal A

genes were introduced via bone-marrow cells, parenterally

or nasally, increased enzyme activity was noted in a range

of organs including the heart and kidneys, along with

reduced GL-3 concentration [8, 9]. In the future, this is

expected to have applications for Fabry disease patients.

(b) Enzyme replacement therapy

It has become possible to create a-Gal A through genetic

engineering methods, for use in treatment of Fabry disease,

and enzyme replacement therapy is becoming a more

standard treatment. In 2001, Eng et al. at Mount Sinai

School of Medicine, and Schiffmann et al. at the US

National Institute of Health had some success with regard

to Fabry disease with enzyme replacement therapy; how-

ever, although a small number of cases demonstrated an

allergic reaction, it was implemented comparatively stably

[10, 11]. The former transferred a-Gal A genes into Chi-

nese hamster ovary cells, while the latter produced a large

volume of enzymes using a gene activation method

involving human fibroblasts, which were used in clinical

trials. Since 2004, enzyme replacement therapy has been

covered by health insurance in Japan, and approximately

260 patients throughout the country are currently receiving

treatment, including dialysis patients.

Sakuraba et al. used methanol-assimilating yeast, which

allows the production of glycoproteins at low cost, to

produce modified human a-Gal A (yr-ha-Gal A). When yr-

ha-Gal A was administered singly to mice with Fabry

disease, a-Gal A activity in various organs increased, and

the increase in a-Gal A activity in the kidney was partic-

ularly notable. Furthermore, yr-ha-Gal A was multiply

administered and the effect on decomposition of GL-3 and

Lyso GL-3, accumulated in the various organs of the mice

with Fabry disease, was confirmed together with the

number of lamellar inclusions [12].

(c) Chemical chaperone treatment

Recently, chemical chaperone treatment using low-

molecular compounds have been attracting attention as a

new method of treating lysosome diseases such as Fabry

disease. This treatment method involves a low-molecular

compound such as a substrate analog, etc., bonding with

the active area of an a-Gal A mutant in a specified amino

acid displacement, correcting the abnormalities in the

enzyme folding, and stabilizing it.

In Fabry disease, 1-deoxygalactonojirimycin and ga-

lactostatin bisulfite, which are a-Gal A substrate analogs,

are known to be effective chaperones. These compounds,

however, are only effective on a-Gal A occurring in

specified amino acid displacements, and have the disad-

vantage of also acting as a-Gal A inhibitors [12, 13].

Recently, the low-molecular drug migalastat hydro-

chloride has also become known as a pharmacological

chaperone that can be administered orally. Pharmacologi-

cal chaperones have the potential to be effective in a wide

range of lysosome conditions, including Fabry disease. At

present, the drug is in Phase III global clinical trials to

confirm its efficacy and effectiveness with regard to Fabry

disease. [14].

(d) Modified a-N-acetyl galactosaminidase with saposin

B treatment

As a new treatment strategy, Sakuraba et al. [15] dis-

placed and modified two amino acids relating to substrate

recognition in a-N-acetyl galactosaminidase (NAGA),

which is similar in three-dimensional structure to a-Gal A.

The modified NAGA acquired the enzyme function of a-Gal

A, while having a greater stability than a-Gal A, and fur-

thermore, included large quantities of M6P, which is related

to absorption into cells. When the modified NAGA was

added to cultured fibroblasts originating from Fabry disease

patients, it was absorbed into the cells and decomposed the

accumulated glycolipids. Modified NAGA did not cause an

immune reaction with blood serum in Fabry disease patients

who demonstrated high antibody value in regard to a-Gal A.

In addition, Sakuraba et al. also developed a manufac-

turing method for saposin B, which strengthens glycolipid

decomposition in a-Gal A and modified NAGA cells.

Saposin B functions to cause encounters between hydro-

philic enzymes and hydrophobic substrates, and its simul-

taneous administration is expected to improve the

effectiveness of the enzyme drug.

Acknowledgments We received a research fund of Japan Kidney

Foundation. We really appreciate Ms Aiko Ooashi for her secretarial

assistance in conducting J-FAST and preparing this manuscript.

Conflict of interest The authors have declared that no Conflict of

interest exists.

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