NDT Advance Access originally published online on October 13, 2006
Nephrology Dialysis Transplantation 2007 22(1):179-186; doi:10.1093/ndt/gfl528
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A nationwide blood spot screening study for Fabry disease in the Czech Republic haemodialysis patient population
tová2
2
avá1
í Motá
411Department of Nephrology, 1st Medical Faculty of Charles University and General Faculty Hospital, 2Institute of Inherited Metabolic Disorders, 1st Medical Faculty of Charles University and General Faculty Hospital, 3Department of Cardiology, 1st Medical Faculty of Charles University and General Faculty Hospital, Prague, 4Institute of Clinical Biochemistry and Laboratory Diagnostics, Charles University School of Medicine and University Hospital, Plzen, 5Nemocnice Hospital Dialysis Centre, Hodonín and 6Medical Faculty Hospital, Masaryk University, Brno, Czech Republic
Correspondence and offprint requests to: Miroslav Merta, MD Department of Nephrology, 1st Medical Faculty of Charles University and General Faculty Hospital, Prague, Czech Republic. Email: merta{at}cesnet.cz
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Abstract |
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 Top Abstract IntroductionSubjects and methodsResultsDiscussionConclusionsAcknowledgementsReferences |
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Background. Fabry disease (FD) is a genetic disorder characterized by accumulation of trihexosylceramide in lysosomes of various tissues leading to multiorgan manifestations, including progressive renal disease. Previous screening studies have shown that a non-neglectable proportion of haemodialysis(HD) patients have unsuspected FD. An extensive FD screening study, the largest to date, has been conducted in HD patients in Czech Republic. We aimed to uncover previously undiagnosed FD patients, to enable them to benefit from cause-specific therapeutic intervention with enzyme replacement therapy (ERT).
Methods. Large-scale screening was executed using a convenient automated enzymatic (
-galactosidose A, -Gal A) dried blood spot on filter paper fluorescence method.
Results. In total, 3370 (45.1% males, 54.9% females) out of 4058 HD patients (83%) in Czech Republic participated in this blood spot screening (BSS) study. Abnormal low fluorescence readings were obtained in 117 patients (3.5%). Subsequent determination of plasma -Gal A activity identified four males and three females with deficient plasma enzyme activity. Determination of -Gal A activity in peripheral blood leucocytes and confirmatory molecular analysis resulted in four newly diagnosed Fabry males and one female. Subsequent family screening identified 10 family members with genotypically proven FD. Based on these screening results, ERT could be offered to five male FD patients.
Conclusions. BSS represents a promising screening tool that has proven to be convenient and effective in uncovering unrecognized FD patients among the chronic HD population in Czech Republic.
Keywords: blood spot screening; end-stage renal disease; enzyme replacement therapy; Fabry disease; -galactosidase A; haemodialysis
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Introduction |
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TopAbstractIntroductionSubjects and methodsResultsDiscussionConclusionsAcknowledgementsReferences |
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Fabry disease (FD) is a rare X-linked genetic lysosomal storage disorder resulting from a defect in the gene encoding lysosomal
-galactosidase A (-Gal A, EC 3.2.1.22
[EC]
). Estimates of the frequency of FD range between 1: 40 000 and 240 000 male individuals [1–3]. The calculated prevalence of FD in Czech population is 1 in 110 000 male live births but the frequency is obviously higher in at risk subpopulations, such as patients requiring chronic haemodialysis (HD).
In FD, the deficiency of -Gal A results in progressive lysosomal accumulation of neutral glycosphingolipids with terminal -galactose moieties, predominantly globotriaosylceramide (Gb3). Accumulation occurs in many cell types, particularly in vascular endothelial cells, renal epithelial cells, pericytes, vascular smooth muscle cells, cardiomyocytes and neurons.
Genetically, the disease is described as an X-linked recessive disorder. Affected hemizygote males have absent or very low levels of lysosomal -Gal A and the majority of these patients will present with the so-called ‘classic’ phenotype with onset of symptoms (angiokeratomas, hypohidrosis, acroparesthesias) during childhood [1]. With advancing age, vascular involvement may lead to end-stage renal disease (ESRD), life-threatening cardiac dysfunction and stroke. Death usually occurs in the third to fifth decade of life. Less frequent phenotypes include the renal and cardiac variants. Patients with the renal variant usually have low plasma -Gal A activity and a milder phenotype, but they may progress to ESRD necessitating HD. In addition, a cardiac variant is seen in males. X-chromosome inactivation in an early stage of embryonic development may (partly) account for the fact that females may be as severely affected as males with FD [4]. However, in women evidence of organ system involvement may be present without overt clinical symptoms.
The gene that encodes -Gal A comprises seven exons and has been isolated, sequenced and localized to the chromosomal region Xq22.1 [5,6]. To date, over 245 mutations of the gene have been identified including non-sense (null) and missense mutations, and splice junction, deletion and insertion errors [7]. Null mutations lead to the complete abolition of the gene transcription into RNA or its translation into functional protein. Active site mutations can reduce enzymatic activity by perturbing the active site while the overall structure of the enzyme remains unaffected. Folding mutations may destabilize the -Gal A protein structure, e.g. by disrupting its hydrophobic core. Since most mutations are private and occur in single FD families, attempts to ascertain strict genotype/phenotype correlations generally have been unsuccessful.
The recent introduction of cause-specific enzyme replacement therapy (ERT) with recombinant -Gal A has renewed the interest in FD, and has initiated several screening studies that aimed to identify ESRD patients with unsuspected FD. The diagnosis of FD in hemizygotes is primarily based on demonstration of deficient -Gal A activity, preferably in leucocytes. In heterozygotes, the diagnosis should be established by mutation testing as enzyme activity may be found within the normal range.
Screening studies that used plasma samples were successful in identifying new FD patients [8,9]. However, for larger-scale screening in high-risk populations management of the logistic burden would be challenging and expensive. The encouraging results, of the first studies that used the FD blood spot screening (BSS) test [10–12], were the stimulus for undertaking a BSS study in Czech Republic that aimed to identify undiagnosed FD patients among the chronic HD patient population. We present the results of this nationwide FD screening study—to date, the largest BSS study that has been conducted. Furthermore, we evaluate the value of the BSS filter paper test as a diagnostic tool in FD.
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Subjects and methods |
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Study design and protocol of BSS study
The nationwide screening for FD among chronic HD patients was performed in all 14 regions of Czech Republic in 2002. The study was coordinated at the Department of Nephrology and the Institute of Inherited Metabolic Disorders of the 1st Medical Faculty of the Charles University and the General Faculty Hospital in Prague, Czech Republic. Approval was obtained from the Ethical Committee of the General Faculty Hospital. All 88 HD centres in Czech Republic were invited to participate in this BSS study, and were supplied with patient information materials, as well as with filter papers to execute the screening test. The local study coordinators at the various HD centres collaborated on the implementation of the BSS study protocol at the investigational sites. HD patients were informed about the BSS study specifics, and those who agreed on participation provided informed consent. Genotyping in confirmed
-Gal A deficient patients was performed with specific written informed consent. The BSS study was designed (i) to test patients with ESRD on chronic HD treatment by means of dried blood spot testing, (ii) to confirm or exclude positive BSS results by measuring -Gal A activity in blood plasma, (iii) to confirm the positive plasma results by determining -Gal A activity in peripheral blood leucocytes and additionally by analysis of urinary Gb3, if urine samples were available (iv) to confirm the diagnosis of FD by molecular analysis of the GLA gene.
Dried blood spot test
Whole blood samples were taken from patients undergoing HD and a few drops were transferred to the BSS filter papers. These were collected, dried at room temperature, stored at 2–8°C and sent for analysis to the Laboratory of the Regina Margherita Children's Hospital in Torino, Italy. The dried blood spots were analysed using a fluorescence-based high throughput microplate method. The BSS test result was defined as positive if the -Gal A activity was found below the threshold of 1.5 nmol/h/ml, which represents approximately 35% of the -Gal A activity in healthy controls. The methodology was based on the results of two published pilot studies [10,11].
-Gal A activity in blood plasma and peripheral blood leukocytes
Plasma was separated from ethylendiaminetetraacetic acid (EDTA) blood (2–3 ml) by centrifugation at 2000 g. Leucocytes were isolated from 5–7 ml EDTA blood using differential dextran sedimentation, erythrocyte lysis and centrifugation at 300 g [13]. Enzyme activities of -Gal A and control enzymes (ß-galactosidase and ß-hexosaminidase) were assayed in plasma samples or in total cell homogenates with artificial 4-methylumbelliferyl (4-MU) substrates [14]. Fluorescence of released 4-MU was measured at excitation 365 and emission 448 nm on the luminescence spectrometer (Perkin Elmer LS50B, Wellesley, USA). Values in leucocytes were expressed as nanomoles (nmol) of substrate degraded per hour per mg of the cell protein; values in plasma as nmol per hour per ml of plasma, at 37°C.
Analysis of glycolipids in urine
Gb3 in urinary sediments was extracted with chloroform-methanol (2:1, v/v) and analysed on HPTLC plates (Silica gel Merck, Germany) [15].
Mutation testing
Genomic DNA and total RNA were extracted from peripheral blood leucocytes or from cultured skin fibroblasts. Genomic DNA was isolated with QiaAmp columns (Qiagen). Total RNA was prepared from white blood cells or cultured skin fibroblasts [16]. Messenger RNA was reverse-transcribed using SuperScriptII reverse transcriptase (Gibco BRL) and oligo(dT)18 according to the protocol supplied by the manufacturer. The sequencing procedure and primer sequences have been described in detail elsewhere [17,18]. In all relatives of identified patients, sequencing was performed both on cDNA and genomic DNA. This approach increased the probability of identifying potential splicing variants caused by mutation and minimize the risk of sequencing only one transcribed allele in female samples (e.g. due to the unbalanced expression of the alleles in tissue).
We also designed the PCR/RFLP or ARMS (Little S) method for further confirmation of the identified mutation, as well as for rapid screening of probands’ family members.
Statistical analyses
Data are shown as mean ± SD.
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Results |
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Study population
In total, 3370 out of 4058 patients (83%) from the overall HD population in Czech Republic participated in this BSS study (Table 1). There were 1521 (45.1%) males and 1849 (54.9%) females. The patients were recruited at 76 out of the 88 (86.3%) Czech HD centres. One male patient who already had been diagnosed with FD underwent BSS testing, but was not additionally tested for enzyme activity or genotyping under this study Protocol.
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BSS test and
-Gal A activity in plasma and peripheral blood leucocytesThe BSS test result was positive (abnormal low fluorescence reading) in 117/3370 enrolled patients (3.5%) of whom 58 (49.6%) were males and 59 (50.4%) females (Table 1). Results of plasma and leucocyte
-Gal A tests are specified in Table 2 per patient and as ranges and means for healthy controls, classic FD controls, cardiac variant FD controls and for a control group of FD heterozygotes. In 7 (four males, three females; Table 1) out of the 117 BSS positive individuals, the plasma -Gal A activity was below the limit set at 3 nmol/h/ml (because females were included in the study, this limit was set higher than the limit normally used in males) (Figure 1). Verification of enzyme deficiency in peripheral blood leucocytes in these seven patients revealed leucocyte -Gal A deficiency in three males (patients 1, 3 and 4) and decreased enzyme activity in male patient 2 and decreased (patient 5) or borderline activity (patient 6 and 7) in three females. The very unusual high residual activity in leucocytes observed in male patient 2 (higher than the activity in patient 1 with a mild cardiac variant, Table 2) will be further investigated. The patient's plasma -Gal A activity was relatively high as well but still indicative of a diagnosis of FD. In the remaining males and females who had a positive BSS test, measurement of -Gal A activity in plasma did not confirm the suspicion of FD.
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Summarizing the results from 117 BSS test positive patients, 4/58 (6.9%) men and 3/59 (5.1%) women had deficient to borderline
-Gal A activity.
Clinical symptoms
At the time of BSS, clinical symptoms that could raise suspicion of FD (i.e. angiokeratoma, hypohidrosis, acroparesthesias, corneal and lenticular opacities and cardiac disease) had not been documented for the seven newly identified FD patients, and their clinical condition had not alerted the respective nephrologists managing their dialysis treatment. All four males and one female patient in whom the diagnosis of FD could be confirmed had been referred and treated under another kidney disease diagnosis than FD (Table 3).
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The illustrative medical histories and clinical presentations for FD patients 2 and 4 are described below to demonstrate the diagnostic pitfalls.
Patient 2
This male patient was diagnosed to have diabetes mellitus type II at age 30 for which he was initiated on insulin therapy 6 years later. Proliferative retinopathy was treated with laser therapy. Further symptoms included ischaemic disease of the heart and lower limbs, arterial hypertension and renal insufficiency presumed to be caused by diabetic nephropathy. Renal disease progressed to chronic renal failure with development of anaemia and hyperuricaemia. Echocardiography revealed discrete signs of left ventricular hypertrophy and pulmonary hypertension. Ejection fraction was 48–50%. At age 45, he started chronic HD treatment, and at that time he tested positive in the BSS and was identified as a previously unrecognized FD patient. At age 46, he underwent a successful kidney transplantation. Graft function was still normal at the most recent examination at the Department of Nephrology in Prague.
The patient's family history was unremarkable (no other siblings and/or off-spring) except a mother who had died at the age of 80 due to pulmonary embolism.
Patient 4
At the age of 16, this male was admitted to the hospital because of complaints of headache. Some pathological urinary findings were detected but not further investigated. At age 26, he was admitted to the district hospital because of headaches and dyspnoea. Examinations revealed arterial hypertension, as well as laboratory signs compatible with advanced renal insufficiency. Renal biopsy was performed at our nephrology department, showing mesangial widening and immunohistological analysis demonstrated slight IgA deposition. Diffuse glomerular sclerosis was compatible with histology seen in end-stage kidney disease. An ultrastructural study was not performed at that time but, at a later stage, the biopsy specimen was re-examined by electron microscopy showing typical signs of FD that had been missed during the initial examination by light microscopy. The patient started chronic HD treatment and shortly thereafter he was found to be positive in the BSS test which led to the diagnosis of FD. Thorough clinical evaluation and taking of a detailed medical history revealed clinical signs and symptoms typical of FD that had been overlooked previously, e.g. hypertrophic cardiomyopathy, polyneuropathy, recurrent fever and pain crises.
The patient's mother who was identified as a heterozygous carrier during family screening had been treated for epilepsia, and one brother who died at age 29 had been receiving chronic HD treatment for 4 years because of ESRD of uncertain aetiology (the patient was a drug abuser). The medical histories of the family members and clinical symptomatology had neither initiated any diagnostic work-up nor had raised suspicion of FD until this BSS study.
Genotyping
In all, four males (mean age 50.4 years, range 26–67) with deficient/decreased -Gal A activity and in one (age 54) out of the three women with decreased/borderline enzyme activity in leucocytes, the mutation in the GLA gene was found (Table 2, Figure 2). Of specific interest, the A143T mutation was detected in male patient 2, i.e. the patient with unusually high residual leucocyte -Gal A activity and disease manifestations apparently limited to the kidney.
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Genetic counselling
The diagnostic work-up in the five diagnosed FD patients included a study of the respective pedigrees which resulted in the identification of several family members with unrecognized FD. The family tree for FD patient 4 (Figure 3) was extremely branched and three additional FD males and four FD females could be uncovered. Shortly after the BSS study was completed and enzyme deficiency had been confirmed, ERT was started in all five male patients from this family; three of them were enrolled into a Fabrazyme® (Genzyme Corporation) clinical study and two were initiated on commercially available Fabrazyme®.
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Discussion |
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TopAbstractIntroductionSubjects and methodsResultsDiscussionConclusionsAcknowledgementsReferences |
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All affected men with FD have absent or reduced activity of
-Gal A which is a prerequisite for using this parameter in the screening for FD among high-risk male patient populations. It is expected that such a screening will identify new FD patients as medical specialists may be unfamiliar with this rare disorder. It is not unusual that diagnosis of individuals with FD symptomatology is delayed for years, or even decades, due to the phenotypic diversity or failure to recognize the constellation of early signs and symptoms. It is illustrative that in one of the patients (patient 4) enrolled in our study, full blown extra-renal FD symptomatology remained unrecognized, and relatively mild FD symptoms did not raise suspicion in four others (patients 1–3 and 5). Patients may initially receive a wrong diagnosis that has to be corrected at a later stage of the disease (e.g. based on analysis of biopsy specimens) when late, multi-systemic, life-threatening complications involving the kidneys, heart and/or central nervous system occur. In some cases with overt symptoms restricted to one organ system, autopsy findings may reveal an unexpected diagnosis of FD [19]. At present, screening in high-risk populations allows newly diagnosed Fabry patients to benefit from the effects of ERT. Indeed, clearance of Gb3 from storage cells and reduction of symptoms have been reported [20–22]. Although fibrotic and sclerotic renal changes cannot be reversed in advanced patients, ERT may still exert a positive impact on major non-renal clinical outcomes [23]. As demonstrated by the pedigree study in the fourth patient's family, genetic counselling likely identifies previously unrecognized FD family members in a relatively early stage of their disease. The outlook for these patients may dramatically improve if ERT is initiated as soon as possible after diagnosis. Thus, early identification of FD patients should be the primary goal of FD screening initiatives.
In two separate Japanese studies and one Dutch study, plasma/whole blood samples were used for quantification of -Gal A activity in cohorts of males with chronic HD. Prevalences of FD of 0.45 [8], 1.2 [9] and 0.22% [24] were calculated. Recently, a new fully automated fluorescence assay was developed by Spada and Pagliardini [11] which enables quantification of residual -Gal A activity in whole blood spots dried on filter paper. In the Italian pilot study, this BSS method detected four FD hemizygotes in a cohort of 1765 (0.22%) male ESRD patients [11]. A nationwide BSS study conducted in Austria enrolled 80.1% of all Austrian chronic HD patients; 85 (3.42%) had a positive BSS result (males 3.50%, females 3.32%) [12]. Four males, of whom three had been diagnosed with FD prior to BSS, had a deficient-Gal A activity in leucocytes and were positively identified as FD patients by molecular analysis.
In the current study, the same BSS method has been used for detection of unrecognized Fabry patients among chronic HD patients in Czech Republic. Our intention to perform a nationwide BSS study was largely fulfilled. In fact, our cohort of 3370 patients (45.1% males, 54.9% females), represented 83% of the chronic HD patient population in 2002. This percentage slightly surpasses the proportion of Austrian HD patients (80.1%) screened for FD [12]. From our cohort of patients who underwent the BSS test, 117 out of 3370 patients (3.5%) tested positive (males 3.81%, females 3.19%). Based on consistently low activity of -Gal A activity in plasma and leucocytes, and result of molecular analysis, overall five new FD patients (4/1521 males, 0.26%; 1/1849 females, 0.05%) could be diagnosed as a result of our BSS study. The proportion of newly diagnosed FD patients in our BSS study largely exceeds the proportion observed in the Austrian BSS study (1/1516 males, 0.07%; 0/964 females, 0%) [12], but is comparable with the frequency observed in the Italian pilot BSS study (4/1765 males, 0.22%) [11] and the Dutch study using whole blood samples (1/508 males, 0.22%). If the male patient, who was already known to have FD and, tested positive in the BSS who is included, the prevalence of FD in Czech HD patients (both sexes combined) reaches 6 out of 3370 patients (0.18%), which compares with the overall prevalence in the Austrian cohort (0.16%). The prevalence of known plus newly diagnosed males in our study was higher; 5 out of 1521 (0.33%) HD patients, compared with 0.26%. In our opinion, the prevalence and other statistical data derived from our BSS study could be considered as representative for Czech Republic HD patient population, and theoretically for other European HD populations, given the large proportion of patients screened.
The sensitivity of BSS in females is markedly lower due to the high percentage of false negative results. Our experience with a large cohort of obligate heterozygotes confirms that there is a considerable overlap between the lower range of normal and -Gal A activity values found in FD heterozygotes (Table 2). In approximately one-third of 21 FD females with a confirmed diagnosis of FD who participated in a recent BSS study in the Netherlands, -Gal A activity was found within the normal range [25]. Therefore, BSS results in females should be interpreted with caution as a substantial proportion of female heterozygotes may remain unidentified in spite of setting broad limits marking the range of ‘suspected’ enzyme activities. Final diagnosis should be established via molecular analysis.
With regard to percentages of new FD patients identified using the BSS test, it can be assumed that only a minor correction could be achieved if the conditions recommended for handling (sampling, drying and storage) of the dried blood spot samples are strictly followed [26]. More favourable BSS testing results can probably be obtained by optimal selection of the population tested—for instance by aiming at patients with renal disease of unknown origin. However, even such a selection is not free of imperfections as illustrated by the fact that the majority of patients diagnosed by BSS had been labelled with some renal disease other than FD prior to enrolment in our BSS study (Table 3). In some, this initial diagnosis was based on renal biopsy analysis which suggests that FD-specific biopsy findings may have been overlooked or were blurred by histological changes due to concomitant renal disease.
The results of molecular analysis differed between male and female patients in our study. In all, four men from the group of seven patients with abnormal or low borderline -Gal A activity, the diagnosis could be confirmed by identification of the GLA mutation. The failure to detect the genetic defect in two females with suspicion of FD seems to be in agreement with the deceiving results of BSS testing in women that were published recently [25]. In the Austrian cohort, there were 11 patients (six males, five females) who had a borderline low leucocyte enzyme activity without a detectable GLA mutation. It has been suggested that the molecular analysis method that was used in the Austrian study (melting point analysis of selected exons) may have missed some GLA mutations [12].
Molecular analysis is unsuitable as an alternative method for large-scale screening in at risk populations as sequencing of the whole GLA gene—a tedious assay—would be required given the uniqueness of FD mutations. Moreover, sequencing is not always conclusive in heterozygotes, as identification of larger deletions in heterozygotes with a wild-type allele may prove to be difficult.
It is of primary importance that the screening method based on enzyme analysis of dry blood spots collected on filter paper bypasses the logistic problems related to transportation of large numbers of whole blood or frozen plasma samples and, additionally, BSS is inexpensive. After storage of the filter papers (up to several weeks) at the recommended temperature, they can be mailed at ambient temperature to a specialized centre [10]. From a laboratory perspective, analysis of blood plasma samples for screening purposes [9] would be technically easier and results would be more accurate. However, storage of large quantities of frozen plasma samples and transportation from distant clinical units to the specialized laboratory is logistically demanding. BSS is far more convenient and safe. Final confirmation in patients with low -Gal A activity in the dry blood spot test and plasma assay requires -Gal activity measurement in cells, typically in peripheral blood leucocytes or lymphocytes, in conjunction with DNA-based mutation analysis [9]. In all patients in our study, plasma -Gal A values corresponded well with values obtained in leucocytes. The high residual activity found in patient 2 suggests that this patient (A143T hemizygote) has a renal Fabry variant. Two patients with the same mutation were found among Italian ESRD patients [11]. High residual -Gal A activity has also been reported in a renal variant patient with the E66Q mutation [9]. These patients lacked the systemic classical manifestations of FD.
The most important issue that evolves from the BSS study presented here is the fact that newly identified patients may be given the opportunity to benefit from cause-specific treatment. The BSS -Gal A activity test and subsequent confirmatory diagnostic procedures enabled us to diagnose five new FD patients, four men and one woman. In addition, many other family members were identified as Fabry patients by pedigree analysis. Between BSS study completion (end 2002) and mid 2005, five male patients have been initiated on ERT and others, who were offered appropriate palliative care are periodically monitored to verify if initiation of ERT is justified and should be proposed to the individual. The expected clinical benefits for newly recognized patients, as well as for affected family members, clearly outweigh the challenges related to such a screening initiative.
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Conclusions |
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TopAbstractIntroductionSubjects and methodsResultsDiscussionConclusionsAcknowledgementsReferences |
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Our nationwide study, the largest to date, provides the rationale for BSS in large cohorts of chronic HD patients to ensure identification of previously unrecognized Fabry patients. As a result of our screening, several of the newly diagnosed FD patients and/or affected family members could be offered effective treatment in the form of ERT. The convenience and safety of the BSS method were confirmed. In the future, FD screening programmes focused on identification of undiagnosed FD patients among high-risk patient populations with kidney disease or other specific extra-renal manifestations compatible with FD (e.g. left ventricular hyperthrophy or early stroke) should be launched. Efforts to increase the awareness of this disease among the medical community should lead to earlier diagnosis of this life-threatening and disabling genetic disease.
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Acknowledgements |
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The study was supported by MSM 0021620810 and by MSM 0021620806.
Conflict of interest statement. None declared.
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Accepted in revised form: 9. 8.06
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