Objectives: Fabry disease is a rare X-linked multisystemic lysosomal storage disorder of the glycosphingolipid metabolic pathway. Nephropathy is one of the most important complications of Fabry disease, and patients with classical phenotype are at risk of developing end-stage kidney disease. In this study, we investigated the use of screening for Fabry disease in kidney transplant recipients at our center.

Materials and Methods: We screened 301 kidney transplant recipients with functioning grafts. Analyses for α-galactosidase A gene mutation were performed in all female and male kidney transplant recipients. We also measured leukocyte α-galactosidase A enzyme activity in patients with identified GLA mutation.

Results: In 301 kidney transplant recipients, mean age was 42.9 ± 12.5 years, and the number of male patients was 180 (60%). Mean time after transplant was 79 ± 56 months, and estimated glomerular filtration rate was 66.8 ± 21 mL/min/1.73 m². One male patient who was diagnosed with Fabry disease before kidney transplant was also evaluated (mutation in the α-galactosidase A gene, c.1093_1101dup [p.Tyr365_lle367dup]). In 2 female patients, p.A143T (c.427G>A) mutation of unknown significance and p.D313Y (c.937G>T) heter­ozygous mutation were identified; however, leukocyte α-galactosidase A enzyme activity was normal in these patients (63.7 and 67.3 nmol/h/mg protein). In the patient diagnosed with Fabry disease, family screening revealed 4 additional affected family members.

Discussions: Although prevalence was shown to be low in our center (1/301 patients; 0.33%), screening studies in kidney transplant recipients may help to detect new patients before they develop life-threatening complications such as renal involvement.

Key words : Family screening, Genetic analysis, Renal transplant recipient

Introduction

Fabry disease (FD) is a rare X-linked multisystemic lysosomal storage disorder of the glycosphingolipid metabolic pathway. Mutations in the α-galactosidase A gene (GLA) encoding the enzyme α-galactosidase A (α-Gal A) are localized on chromosome Xq22. Mutations cause accumulation of glycosphingolipids in a wide variety of cells, especially the intracellular accumulation of globotriaosylceramide (Gb3), which can result in renal, cardiac, and cerebrovascular diseases leading to progressive organ failure.¹ To date, over 900 GLA mutations have been identified,² but not every GLA mutation is pathogenic, and most mutations are specific to a family. Therefore, there are no clear genotype and phenotype correlations, and the course of the disease is variable among individuals.^3,4

Although FD occurs in all ethnic groups and affects predominantly males, carrier (heterozygous) women may be mildly or severely affected.⁵ The incidence in males is 1:50 000 and ranges from 1:40 000 to 1:117 000 in different populations.^6,7 With the consideration of factors such as the rare occurrence of the disease, nonspecific symptoms in early stages, different variants, lack of awareness among clinicians, and initial misdiagnosis and/or delays in diagnosis, the actual prevalence is probably higher than shown in the available data.^8,9 In particular, the prevalence seems to be higher among high-risk patients with left ventricular hypertrophy, premature stroke, and renal replacement therapy.¹⁰

Nephropathy is one of the most important complications of FD, and patients with classical phenotype are at risk of developing end-stage kidney disease (ESKD) between the third and fifth decades of life.^11,12 Studies on FD screening in hemodialysis patients have shown the prevalence to be between 0.02% and 1.2%.¹³ In Turkey, the prevalence of FD was found to be between 0.12% and 0.95%^14,15 in cohorts of patients with selected chronic kidney diseases (CKD) and between 0.17% and 0.3% in patients on hemodialysis.^16,17 Differences between prevalences are explained by the size of the screened population, male/female patient selection, screening methods, and disease definition (ie, classical or variant).¹³

Few selective screening studies are available on kidney transplant recipients. In addition, the number of patients diagnosed varies between different countries and regions.^18-21 It is important to investigate the primary cause of kidney disease in this high-risk population. Early verification of FD can allow more efficient management of patients and lead to screening of other family members and appropriate genetic counseling. In this context, we studied FD screening in patients with kidney transplant at our center.

Materials and Methods

Study design and patient selection
Kidney transplant recipients (> 18 years) followed up at Ankara University School of Medicine, Transplantation Center (Ankara, Turkey) were invited to participate in this study between May 2017 and June 2018. Patients with known causes of renal failure were also included. In total, 301 kidney transplant recipients with functioning grafts were evaluated. Genetic analyses were performed in all female and male patients as a screening test.

This study was approved by the Ankara University School of Medicine Ethics Committee for Clinical Studies in accordance with Helsinki Declaration guidelines. Written informed consent was obtained from all participants.

Data collection
Clinical, laboratory, and demographic data of all patients were obtained from patient medical files and the hospital database at the time of screening. Demographic features (age and biologic sex) and clinical and laboratory characteristics were recorded. Estimated glomerular filtration rate was calculated by using the Chronic Kidney Disease Epidemiology Collaboration creatinine equation.²²

Analysis for GLA mutation was performed to confirm the diagnosis of FD in all patients as an initial diagnostic assay. We also measured α-Gal A enzyme activity in patients with identified GLA mutation. In patients with GLA mutation, detailed medical and family histories, as well as physical examinations, were also obtained. In addition, cardiac, neurologic, dermatologic, and ophthal­mologic investigations were performed in terms of the signs and symptoms associated with FD in these patients.

Laboratory analyses
Determination of α-Gal A enzyme activity was performed by using the dried blood spot technique, which was previously described by Chamoles and associates.23 Blood was spotted directly on the filter paper after lancet finger prick or venipuncture syringe draw. The entire circle was uniformly saturated. Cards were air-dried for at least 3 hours and stored in sealed plastic bags at 4°C for up to 1 week in a cabinet and at -20°C with a desiccant and a humidity indicator if longer periods of storage were needed.

In patients with a positive dried blood spot screening test, α-Gal A enzyme activity was examined again or peripheral white blood cells (reference range: > 23.1 nmol/h/mg protein) were further examined at the Duzen Laboratory Group (Ankara, Turkey).²⁴

α-Galactosidase A enzyme activity in white blood cells
Analysis of α-Gal A enzyme activity was performed using the fluorimetric method. 4-Methylumbelliferyl α-D-galactopyranoside (no. M334475; Toronto Research Chemicals, Toronto, Canada) was used as substrate, and N-acetyl-D-galactosamine (no. A2795; Sigma-Aldrich, St. Louis, MO, USA) was used as inhibitor. Incubation was carried out with 3-mm dried blood spot punch, inhibitor, and substrate at 37°C for 17 hours. Fluorescent molecules were labeled at 366-nm excitation and 442-nm emission in fluorimetry (BioTek Synergy, Winooski, VT, USA). Measurements were evaluated using the calibration curve of 4-methylumbelliferone (no. M1381; Sigma-Aldrich).

α-Galactosidase A mutation analysis
Genetic analysis for mutation in GLA was performed at the Intergen Genetic Diagnosis Center (Ankara, Turkey). Sequence analyses for GLA were conducted with the use of MiSeq next-generation sequencing platform, a US Food and Drug Administration-approved diagnostic system (Illumina, Inc., San Diego, CA, USA). All coding exons of the gene and their flanking splice site junctions were amplified using polymerase chain reaction primers, designed with PRIMER Designer software version 2.0 (Scientific & Educational Software, Westminster, CO, USA). Libraries were prepared with the Nextera XT kit (Illumina, Inc.) according to the manufacturer’s instructions. Next-gene sequencing was carried out on MiSeq (Illumina, Inc.). Sequences were aligned to the hg19 genome within MiSeq Reporter software (Illumina, Inc.). Visualization of the data was performed with IGV 2.3 software (Broad Institute, Cambridge, MA, USA).

Results

We screened 301 kidney transplant recipients for FD. A genetic analysis of 1 male patient who was diagnosed with FD before kidney transplant confirmed the diagnosis of FD and showed a previously reported hemizygous variant in the GLA (c.1093_1101dup [p.Tyr365_lle367dup]). We also identified GLA mutations in 2 female patients. In each of these patients, the p.A143T (c.427G>A) mutation of unknown significance and the p.D313Y (c.937G>T) heterozygous mutation were identified. However, leukocyte α-Gal A enzyme activity was normal in these 2 patients (63.7 and 67.3 nmol/h/mg protein, respectively).

At the time of screening, average age was 42.9 ± 12.5 years (range, 18-79 y), and the number of male patients was 180 (60%). Mean time after transplant was 79 ± 56 months, and estimated glomerular filtration rate was 66.8 ± 21 mL/min/1.73 m2. The general characteristics of the study population are shown in Table 1.

Case 1
A 42-year-old female patient received living-donor kidney transplant at the age of 27 years. The cause of primary kidney disease was unknown. Her graft function was stable (serum creatinine level of 0.7 mg/dL and no proteinuria or hematuria). She had hypertension and was taking carvedilol medication. Her echocardiogram showed left ventricular hypertrophy. No signs and symptoms of retinal, corneal, cutaneous, and neurologic complications associated with FD were found. She had no family history of kidney disease. Genetic analysis detected p.A143T (c.427G>A) mutation. The patient’s leukocyte α-Gal A enzyme activity was 63.7 nmol/h/mg protein. She was not scheduled for enzyme replace­ment therapy (ERT).

Case 2
This female patient was 52 years old. The cause of primary kidney disease was unknown. She received a kidney transplant from a deceased donor 5 years previously. Her creatinine level was 1 mg/dL, and she had no proteinuria and hematuria. Comorbidities included hypertension and epilepsy. She was taking carvedilol and valproic acid. There was no history of kidney disease or signs and symptoms compatible with FD in her family. A physical examination did not reveal any clinical signs of FD. Her genetic testing showed the p.D313Y (c.937G>T) heterozygous mutation. Her leukocyte α-Gal A enzyme activity was 67.3 nmol/h/mg protein. Detailed clinical and laboratory investigations concluded that she did not have FD. Therefore, ERT was not indicated.

Case with Fabry disease
The case with FD was a 37-year-old male patient. He was admitted to our center for a kidney transplant from his father. The patient was on hemodialysis for 1 year. Physical examination of the patient revealed angiokeratoma. Echocardiography showed left ventricular hypertrophy. He did not have a history of ischemic coronary artery disease or stroke. However, he had family history of kidney disease, and his brother was on maintenance hemodialysis. Due to the clinical suspicion attributed to FD, GLA mutation analysis and enzyme activity tests were performed. Genetic analysis of the patient confirmed the diagnosis of FD and showed the previously reported hemizygous variant in the GLA (c.1093_1101dup [p.Tyr365_lle367dup]). His leukocyte α-Gal A enzyme activity was low (1.4 nmol/h/mg protein). We performed screening of other family members (n = 11 family members). The patient’s mother and his 3 daughters were detected to carry the heterozygous mutation. The patient had 2 brothers (36 years and 16 years) and 1 sister (26 years). However, the c.1093_1101dup (p.Tyr365_lle367dup) mutation was only detected in the 36-year-old brother. This brother also had ESKD, left ventricular hypertrophy, angiokeratoma, and low leukocyte α-Gal A enzyme activity (3.7 nmol/h/mg protein). The pedigree of the patient is depicted in Figure 1. After the family was screened, he received a kidney graft from his father. After transplant, his graft function has remained stable (serum creatinine level of 1.7 mg/dL and no proteinuria) and he has received standard immunosuppressive therapy.

The patient and his brother are currently receiving ERT.

Discussion

In this study, we examined FD screening in kidney transplant recipients at our center. A GLA variant or mutation was detected in 2 female patients. However, leukocyte α-Gal A enzyme activity was found to be normal in these 2 patients. After a detailed medical and family history of these patients was conducted, it was concluded that there was no FD based on medical review and clinical and laboratory data. Therefore, ERT was not planned. We also evaluated 1 male patient diagnosed with FD before kidney transplant. Family screening revealed affected relatives. For this patient diagnosed with FD, 4 additional affected family members were identified.

In the past decade, despite increased patho­physiology studies of the disease, information on its course among individuals, and information about its treatment, most patients are still misdiagnosed or diagnosed late.¹³ This is notable because screening studies performed in patients undergoing dialysis treatment and with kidney transplant have shown a high prevalence of FD.^16-21 Therefore, it is important to investigate the cause of primary kidney disease in this high-risk population; a definitive diagnosis in these patients will prevent the complications that may occur and will lead to the identification of the disease in other family members.¹³

The European Renal Best Practice guideline recommends FD screening in men under 50 years of age and in unexplained CKD in women of any age. For FD, plasma and/or leukocyte α-Gal A enzyme activity deficiency should be determined for all homozygous men, with GLA analysis performed to confirm FD and to identify mutations. In heterozygous women, due to random X-chromosome inactivation, usually slightly reduced or normal enzyme activity is observed. Therefore, the demonstration of GLA mutation in women is mandatory for the diagnosis of FD.²⁵ In our study, GLA mutation analysis was performed in all patients as the initial diagnostic assay. We then determined the level of enzyme activity in patients with positive GLA mutation. When α-Gal A enzyme activity is normal, this indicates that patients are not likely carriers of FD.

Fabry disease is manifested by variable clinical phenotypes. Three different variant phenotypes (classical, cardiac, and kidney) have been described. The classical phenotype is usually symptomatic in childhood or adolescence and is characterized by angiokeratoma, hypohidrosis, corneal and lenticular opacities, acroparesthesia, abdominal pain, and hearing disorders. Kidney disease usually begins with microalbuminuria and proteinuria in the second and third decades of life, and its severity increases with age. End-stage kidney disease usually occurs between the third and fifth decades of life. Cardiac involvement typical occurs before the age of 30 years and is characterized by left ventricular hypertrophy, arrhythmias, cardiomyopathy, valvular abnormalities, and premature coronary artery disease. Transient ischemic attack and ischemic stroke are common and depend on small vessel events. Life expectancy for those with the classical phenotype is reduced by 20 years due to life-threatening ESKD and cardiovascular and/or cerebrovascular complications.¹ In our study, the cause of primary kidney disease in patients with positive mutation analysis was unknown. After a detailed clinical examination in 2 female patients, we did not detect any clinical signs associated with FD.

In studies in which patients with kidney transplant are screened, different GLA mutations and variants have been identified. By the interpretation of their clinical significance, the characteristic features of FD, and laboratory and clinical characteristics of patients, the prevalence can be calculated. In a study involving 673 kidney transplant patients in Belgium, pathogenic missense mutation (p.A143T) was identified in only 1 male patient.¹⁸ In a nationwide study in Austria in which 1306 male kidney transplant patients were screened, 2 new patients were diagnosed with FD.¹⁹ In Turkey, in the study from Yılmaz and colleagues, in which 1095 kidney transplant patients (648 women, 447 men) were included, genetic variations related to FD were identified in 8 patients (5 men, 3 women).20 In another recent multicentered study that included 3822 kidney transplant patients in Turkey, pathogenic GLA mutation was identified in 15 patients.²¹ In our study, 301 kidney transplant recipients were screened, and A143T and D313Y variants/mutations were detected in 2 female patients.

The clinical significance of the A143T variant in GLA is a matter of debate. In the literature, this variant has clinical effects ranging from symptoms and findings of classical FD to healthy presentation. In addition, as newborn screening studies for FD increase, more people with this variant will be identified, and this is forcing clinicians to consider ways to manage the disease in these individuals.^26-29 Similarly, there is no clear consensus on the pathogenesis of the D313Y variant/mutation. The D313Y mutation is a change that results in a 60% reduction in enzyme activity (stable in lysosomal pH but reported to decrease activity in neutral pH) and is defined as pseu­dodeficiency allele.^30,31 Most clinical studies support that the D313Y mutation is not pathogenic. In a study from Oder and associates, individuals with D313Y genotype did not present with serious organ damage in long-term follow-up. In this study, leukocyte α-Gal A enzyme activity and serum lysosomal Gb3 levels were normal in all patients. Serum lysosomal Gb3 levels, which play an important role in the pathogenicity of FD, is therefore considered a biomarker for disease severity and prognosis.³²

In studies involving high-risk patients, D313Y mutation has been detected in 3 of 493 patients with stroke diagnosis,33 in 3 of 508 patients with hypertrophic cardiomyopathy,34 in 2 of 1453 patients with CKD,¹⁵ and in 2 of 911 patients on hemodialysis.³⁵ Lenders and colleagues, in an examination of D313Y mutations found in 7 people in a family of 9, suggested a relationship between D313Y mutation and white matter lesions. They reported that they observed white matter lesions in all 7 people with mutations in this family; however, the 2 individuals without mutations did not have these lesions.³⁶

In Turkey, in studies that included patients with kidney transplant, D313Y mutation was reported in 2 of 1095 kidney transplant recipients²⁰ and in 19 of 3822 kidney transplant recipients.²¹ However, none of these patients had symptoms and findings specific to FD. In our study, D313Y mutation was detected in 1 of 301 patients. Leukocyte α-Gal A enzyme activity was normal in this patient. As a result of detailed evaluation of the patient, no symptoms and/or findings specific to FD were found.

It is not clear whether ERT can be used in patients with kidney transplant. The European Renal Best Practice guideline does not recommend ERT for renal indications in FD after kidney transplant. However, in patients who receive ERT because of nonrenal indications, it is recommended to continue treatment even after transplant.²⁵

Conclusions

Early diagnosis of FD and increased awareness among clinicians about FD and its complications are important for the prevention of progressive organ damage. The results of genetic mutations in patients with weak genotype-phenotype correlations may cause controversy with regard to use of ERT. Detailed medical and family histories, as well as physical examinations, are essential in these patients. Beyond an unnecessary treatment, it is important to recognize that misdiagnosis can bring an emotional burden on patients and their families, which may adversely affect their quality of life.

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