Clin Exp Nephrol DOI 10.1007/s10157-013-0897-2
REVIEW ARTICLE
WCN 2013 Satellite Symposium ‘‘Kidney and Lipids’’
Fabry disease: experience of screening dialysis patients for Fabry disease Eiji Kusano • Osamu Saito • Tetsu Akimoto Yasushi Asano
•
Received: 25 September 2013 / Accepted: 18 October 2013 Ó Japanese Society of Nephrology 2013
Abstract The prevalence rate for Fabry disease is conventionally 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 (aGal 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 polymorphism. 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
E. Kusano (&) Utsunomiya Social Insurance Hospital, 11-17, Minami Takasago, Utsunomiya, Tochigi 321-0143, Japan e-mail: [email protected] E. Kusano Jichi Medical University, Shimotsuke, Japan
pharmacological chaperone, but is currently in Phase III global clinical trials. Adding saposin B to modified a-Nacetyl 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 globotriosylceramide (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.
O. Saito T. Akimoto Division of Nephrology, Department of Medicine, Jichi Medical University, Shimotsuke, Japan
Fabry disease subtype
Y. Asano Koga Red Cross Hospital, Jichi Medical University, Shimotsuke, Japan
The condition known as Fabry disease was first reported by the British and German dermatologists, Anderson and
123
Clin Exp Nephrol
Fabry, in 1898. They noted the characteristic dermatological 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 chromosome 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 accumulating in tissue and organs, causing symptoms in the skin, eyes, digestive organs, heart and kidneys, as well as psychological 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 hypohidrosis, angiokeratoma, corneal clouding and other symptoms. With increasing age, the patient may suffer endstage 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 classical 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 hypertrophic cardiomyopathy. The mechanisms by which subtypes other than classical type occur are extremely interesting; it has been conjectured to date that mutations without particular 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 frequency of the condition is higher than was previously
123
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 easiest, 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 disease, 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 implement 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 demonstrated 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 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
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 testing. Alternatively, since the a-Gal A substrate globotriaosylsphingosine (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 effectiveness 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 polymorphism. 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, pathological 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 electron 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
123
Clin Exp Nephrol
patients develop ESKD they are treated with dialysis and/ or renal transplantation. Thrombosis is treated with ticlopidine 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; however, 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 Chinese 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 yrha-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 particularly 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 lowmolecular 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
123
acid displacement, correcting the abnormalities in the enzyme folding, and stabilizing it. In Fabry disease, 1-deoxygalactonojirimycin and galactostatin 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 disadvantage of also acting as a-Gal A inhibitors [12, 13]. Recently, the low-molecular drug migalastat hydrochloride has also become known as a pharmacological chaperone that can be administered orally. Pharmacological 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] displaced 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 furthermore, 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 manufacturing method for saposin B, which strengthens glycolipid decomposition in a-Gal A and modified NAGA cells. Saposin B functions to cause encounters between hydrophilic enzymes and hydrophobic substrates, and its simultaneous 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 interest exists.
The authors have declared that no Conflict of
References 1. Doi K, Noiri E, Ishizu T, Negishi K, Suzuki Y, Hamasaki Y, et al. High-throughput screening identified disease-causing mutants and functional variants of a-galactosidase A gene in Japanese male hemodialysis patients. J Hum Genet. 2012;57:575–9.
Clin Exp Nephrol 2. Spada M, Pagliardini S, Yasuda M, Tukel T, Thiagarajan G, Sakuraba H, et al. High incidence of later-onset fabry disease revealed by newborn screening. Am J Hum Genet. 2006;79(1): 31–40. 3. Hwu WL, Chien YH, Lee NC, Chiang SC, Dobrovolny R, Huang AC, et al. Newborn screening for Fabry disease in Taiwan reveals a high incidence of the later-onset GLA mutation c.936 ? 919G [ A (IVS4 ? 919G [ A). Hum Mutat. 2009;30(10): 1397–405. 4. The state of chronic dialysis therapy in Japan (as of 31st December 2010). J Jpn Soc Dial Ther 2012;45:1–47. 5. Auray-Blais C, Ntwari A, Clarke JT, Warnock DG, Oliveira JP, Young SP, et al. How well does urinary lyso-Gb3 function as a biomarker in Fabry disease? Clin Chim Acta. 2010;411 (23–24):1906–14. 6. Togawa T, Kodama T, Suzuki T, Sugawara K, Tsukimura T, Ohashi T, et al. Plasma globotriaosylsphingosine as a biomarker of Fabry disease. Mol Genet Metab. 2010;100(3):257–61. 7. Kobayashi M, Ohashi T, Fukuda T, Yanagisawa T, Inomata T, Nagaoka T, et al. No accumulation of globotriaosylceramide in the heart of a patient with the E66Q mutation in the a-galactosidase A gene. Mol Genet Metab. 2012;107:711–5. 8. Takenaka T, Murray GJ, Qin G, Quirk JM, Ohshima T, Qasba P, et al. Long-term enzyme correction and lipid reduction in multiple organs of primary and secondary transplanted Fabry mice receiving transduced bone marrow cells. Proc Natl Acad Sci USA. 2000;97(13):7515–20.
9. Jung SC, Han IP, Limaye A, Xu R, Gelderman MP, Zerfas P, et al. Adeno-associated viral vector-mediated gene transfer results in long-term enzymatic and functional correction in multiple organs of Fabry mice. Proc Natl Acad Sci USA. 2001;98(5):2676–81. 10. Eng CM, Guffon N, Wilcox WR, Germain DP, Lee P, Waldek S, et al. International collaborative Fabry disease study group. Safety and efficacy of recombinant human alpha-galactosidase A–replacement therapy in Fabry’s disease. N Engl J Med. 2001;345(1):9–16. 11. Schiffmann R, Kopp JB, Austin HA 3rd, Sabnis S, Moore DF, Weibel T, et al. Enzyme replacement therapy in Fabry disease: a randomized controlled trial. JAMA. 2001;285(21):2743–9. 12. Tsukimura T, Kawashima I, Togawa T, Kodama T, Suzuki T, Watanabe T, et al. Efficient uptake of recombinant a-galactosidase A produced with a gene-manipulated yeast by Fabry mice kidneys. Mol Med. 2012;10(18):76–82. 13. Tsukimura T, Chiba Y, Ohno K, Saito S, Tajima Y, Sakuraba H. Molecular mechanism for stabilization of a mutant a-galactosidase A involving M51I amino acid substitution by imino sugars. Mol Genet Metab. 2011;103(1):26–32. 14. http://www.amicustherapeutics.com/default.asp. 15. Tajima Y, Kawashima I, Tsukimura T, Sugawara K, Kuroda M, Suzuki T, et al. Use of a modified alpha-N-acetylgalactosaminidase in the development of enzyme replacement therapy for Fabry disease. Am J Hum Genet. 2009;85(5):569–80.
123
REVIEW ARTICLE
WCN 2013 Satellite Symposium ‘‘Kidney and Lipids’’
Fabry disease: experience of screening dialysis patients for Fabry disease Eiji Kusano • Osamu Saito • Tetsu Akimoto Yasushi Asano
•
Received: 25 September 2013 / Accepted: 18 October 2013 Ó Japanese Society of Nephrology 2013
Abstract The prevalence rate for Fabry disease is conventionally 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 (aGal 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 polymorphism. 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
E. Kusano (&) Utsunomiya Social Insurance Hospital, 11-17, Minami Takasago, Utsunomiya, Tochigi 321-0143, Japan e-mail: [email protected] E. Kusano Jichi Medical University, Shimotsuke, Japan
pharmacological chaperone, but is currently in Phase III global clinical trials. Adding saposin B to modified a-Nacetyl 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 globotriosylceramide (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.
O. Saito T. Akimoto Division of Nephrology, Department of Medicine, Jichi Medical University, Shimotsuke, Japan
Fabry disease subtype
Y. Asano Koga Red Cross Hospital, Jichi Medical University, Shimotsuke, Japan
The condition known as Fabry disease was first reported by the British and German dermatologists, Anderson and
123
Clin Exp Nephrol
Fabry, in 1898. They noted the characteristic dermatological 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 chromosome 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 accumulating in tissue and organs, causing symptoms in the skin, eyes, digestive organs, heart and kidneys, as well as psychological 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 hypohidrosis, angiokeratoma, corneal clouding and other symptoms. With increasing age, the patient may suffer endstage 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 classical 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 hypertrophic cardiomyopathy. The mechanisms by which subtypes other than classical type occur are extremely interesting; it has been conjectured to date that mutations without particular 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 frequency of the condition is higher than was previously
123
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 easiest, 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 disease, 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 implement 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 demonstrated 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 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
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 testing. Alternatively, since the a-Gal A substrate globotriaosylsphingosine (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 effectiveness 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 polymorphism. 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, pathological 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 electron 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
123
Clin Exp Nephrol
patients develop ESKD they are treated with dialysis and/ or renal transplantation. Thrombosis is treated with ticlopidine 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; however, 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 Chinese 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 yrha-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 particularly 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 lowmolecular 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
123
acid displacement, correcting the abnormalities in the enzyme folding, and stabilizing it. In Fabry disease, 1-deoxygalactonojirimycin and galactostatin 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 disadvantage of also acting as a-Gal A inhibitors [12, 13]. Recently, the low-molecular drug migalastat hydrochloride has also become known as a pharmacological chaperone that can be administered orally. Pharmacological 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] displaced 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 furthermore, 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 manufacturing method for saposin B, which strengthens glycolipid decomposition in a-Gal A and modified NAGA cells. Saposin B functions to cause encounters between hydrophilic enzymes and hydrophobic substrates, and its simultaneous 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 interest exists.
The authors have declared that no Conflict of
References 1. Doi K, Noiri E, Ishizu T, Negishi K, Suzuki Y, Hamasaki Y, et al. High-throughput screening identified disease-causing mutants and functional variants of a-galactosidase A gene in Japanese male hemodialysis patients. J Hum Genet. 2012;57:575–9.
Clin Exp Nephrol 2. Spada M, Pagliardini S, Yasuda M, Tukel T, Thiagarajan G, Sakuraba H, et al. High incidence of later-onset fabry disease revealed by newborn screening. Am J Hum Genet. 2006;79(1): 31–40. 3. Hwu WL, Chien YH, Lee NC, Chiang SC, Dobrovolny R, Huang AC, et al. Newborn screening for Fabry disease in Taiwan reveals a high incidence of the later-onset GLA mutation c.936 ? 919G [ A (IVS4 ? 919G [ A). Hum Mutat. 2009;30(10): 1397–405. 4. The state of chronic dialysis therapy in Japan (as of 31st December 2010). J Jpn Soc Dial Ther 2012;45:1–47. 5. Auray-Blais C, Ntwari A, Clarke JT, Warnock DG, Oliveira JP, Young SP, et al. How well does urinary lyso-Gb3 function as a biomarker in Fabry disease? Clin Chim Acta. 2010;411 (23–24):1906–14. 6. Togawa T, Kodama T, Suzuki T, Sugawara K, Tsukimura T, Ohashi T, et al. Plasma globotriaosylsphingosine as a biomarker of Fabry disease. Mol Genet Metab. 2010;100(3):257–61. 7. Kobayashi M, Ohashi T, Fukuda T, Yanagisawa T, Inomata T, Nagaoka T, et al. No accumulation of globotriaosylceramide in the heart of a patient with the E66Q mutation in the a-galactosidase A gene. Mol Genet Metab. 2012;107:711–5. 8. Takenaka T, Murray GJ, Qin G, Quirk JM, Ohshima T, Qasba P, et al. Long-term enzyme correction and lipid reduction in multiple organs of primary and secondary transplanted Fabry mice receiving transduced bone marrow cells. Proc Natl Acad Sci USA. 2000;97(13):7515–20.
9. Jung SC, Han IP, Limaye A, Xu R, Gelderman MP, Zerfas P, et al. Adeno-associated viral vector-mediated gene transfer results in long-term enzymatic and functional correction in multiple organs of Fabry mice. Proc Natl Acad Sci USA. 2001;98(5):2676–81. 10. Eng CM, Guffon N, Wilcox WR, Germain DP, Lee P, Waldek S, et al. International collaborative Fabry disease study group. Safety and efficacy of recombinant human alpha-galactosidase A–replacement therapy in Fabry’s disease. N Engl J Med. 2001;345(1):9–16. 11. Schiffmann R, Kopp JB, Austin HA 3rd, Sabnis S, Moore DF, Weibel T, et al. Enzyme replacement therapy in Fabry disease: a randomized controlled trial. JAMA. 2001;285(21):2743–9. 12. Tsukimura T, Kawashima I, Togawa T, Kodama T, Suzuki T, Watanabe T, et al. Efficient uptake of recombinant a-galactosidase A produced with a gene-manipulated yeast by Fabry mice kidneys. Mol Med. 2012;10(18):76–82. 13. Tsukimura T, Chiba Y, Ohno K, Saito S, Tajima Y, Sakuraba H. Molecular mechanism for stabilization of a mutant a-galactosidase A involving M51I amino acid substitution by imino sugars. Mol Genet Metab. 2011;103(1):26–32. 14. http://www.amicustherapeutics.com/default.asp. 15. Tajima Y, Kawashima I, Tsukimura T, Sugawara K, Kuroda M, Suzuki T, et al. Use of a modified alpha-N-acetylgalactosaminidase in the development of enzyme replacement therapy for Fabry disease. Am J Hum Genet. 2009;85(5):569–80.
123
