The Relationship of Vascular Glycolipid Storage to Clinical ... : Medicine (original) (raw)
INTRODUCTION
Fabry disease is a rare X-linked lysosomal storage disorder caused by deficiency of α-galactosidase A (α-Gal A) activity, the lysosomal enzyme that breaks down globotriaosylceramide (Gb3, also known as GL-3) and other glycosphingolipids3,7,8. Deficient α-Gal A activity leads to accumulation of Gb3 in most tissues in the body, with life-threatening clinical consequences in the kidney, heart, and cerebrovascular system.
Although few heterozygous females have the classic Fabry phenotype, there is increasing evidence that many carriers have isolated or multiple manifestations of Fabry disease, such as left ventricular hypertrophy, valvular disease, abnormal renal function, neuropathic pain, and chronic gastrointestinal symptoms10,11,14,21,28-30. These manifestations are presumably due to the normal process of random X-chromosomal inactivation (lyonization) during embryogenesis resulting in varying proportions of cells containing the normal X chromosome and cells containing the X-chromosome with the α-Gal A mutation2.
Classically affected males have less than 1% normal α-Gal A activity and an estimated median age at death of 50-55 years4,14 (an increase of 10-15 years6 since the advent of renal dialysis and transplantation). Heterozygous females with Fabry disease typically have low to normal α-Gal A activity, later onset of symptoms, and disease manifestations that are less acute and more isolated (that is, confined to 1 organ system), although some women have symptoms as severe as those of classically affected males27. An analysis of the United States Renal Disease System database determined that 12% of the Fabry patients on dialysis from 1995 to 1998 were female24. However, overall very little is known about the true frequency and extent of disease penetrance in women. No large-scale prospective studies of the disease in women or men have been performed due to its rarity, and most of the literature focuses on Fabry disease in men.
Enzyme replacement therapy for Fabry disease became available in Europe in 2001 with agalsidase alfa (Replagal, Transkaryotic Therapies Inc, Cambridge MA) and agalsidase beta (Fabrazyme, Genzyme Corp, Cambridge, MA) and in the United States in 2003 with agalsidase beta only (Fabrazyme, Genzyme Corp, Cambridge, MA) following randomized controlled clinical trials7,8,22. Although long-term efficacy remains to be demonstrated, the availability of this disease-specific therapy represents an important advance for Fabry patients, who formerly had only nonspecific and palliative treatment options (for example, renal dialysis and transplant) that can prolong life but do not address the underlying cause of the disease. With the availability of enzyme replacement therapy, prompt diagnosis and treatment have a new importance. Typically, while patients are on enzyme replacement therapy, their neurologic, cardiac, and renal status is followed carefully.
We conducted this prospectively designed study to collect normative data on women with Fabry disease of varying severity to better understand how the disease manifests in women and to identify appropriate endpoints for future clinical trials of enzyme replacement therapy in women.
PATIENTS AND METHODS
No treatment was administered in this prospectively designed, single-center study. Participants had to be 18 years of age or older, with signs or symptoms consistent with Fabry disease and no prior treatment with α-Gal A enzyme replacement therapy. Patients were referred to the study by their primary care physicians or were family members of patients enrolled in other Fabry disease studies at the National Institutes of Health. The Institutional Review Board approved the study, and all patients gave their written informed consent before participating in the study. Women who had used an investigational drug within 30 days of study enrollment, had diabetic neuropathy or other confounding renal disorder, had clinically significant organic disease, or had an unstable condition that in the opinion of the investigator should preclude study participation were excluded from the study. Women of childbearing age agreed to a pregnancy test; patients who were pregnant or lactating were allowed to participate but were excluded from the magnetic resonance imaging (MRI) and magnetic resonance angiography (MRA) procedures.
Data from participants were collected over a 7-day period. Patients were asked to discontinue all pain medications, including prophylactic neuropathic pain medications (antiepileptics and narcotics), 7 days before beginning the study and for the duration of the study, although if needed, they could be resumed as rescue medications during the study. On day 1 of the study, medical histories were taken and all laboratory testing and evaluations were performed. During the 7-day study, patients recorded use of rescue pain medications daily in a pain diary. On day 7 (or right before pain medications were resumed), patients filled out the Brief Pain Inventory (BPI) questionnaire. Study evaluations included the following: obtaining a detailed medical and surgical history; physical examination including physician assessment of Fabry symptoms; neurologic assessment; ophthalmologic examination (including slit lamp exam); cardiac assessment (including electrocardiogram and echocardiogram); skin biopsy (for Gb3 content); routine blood chemistry, hematology, and urinalysis; renal function tests including creatinine clearance, estimated glomerular filtration rate (GFR), and urinary protein excretion; cerebrovascular evaluation (head MRI/MRA); Fabry-specific blood tests (genotyping, plasma Gb3, plasma and leukocyte α-Gal A activity), and urine test (urinary Gb3 excretion). Genotyping was performed by standard polymerase chain reaction (PCR) and sequencing techniques1. Briefly, all samples were genotyped at Genzyme by the Clinical Laboratory Science Unit. Seven PCR products covering the 7 exons and at least 100 bp of flanking intronic sequence of the α-Gal A gene were amplified with gene-specific primers (R. Pomponio, personal communication). PCR products were prepared and sequenced on an Applied Biosystems 3100 Automated DNA sequencer using the ABI BigDye V3 chemistry according to the manufacturer's recommendations. Sequence analysis comparison was performed using the Sequencher v 3.3.1 Software (GeneCodes Corp, Ann Arbor, MI). Gb3 was measured in plasma by mass spectroscopy20 and in urine by high performance liquid chromatography as described26. Gb3 was measured in skin by light microscopic evaluation. Punch biopsies were obtained and prepared as described previously25. Dermal capillary endothelial cells were scored using a none-mild-moderate-severe accumulation scale of 0-1-2-3. Activity of α-Gal A was measured using a fluorometric assay. GFR was estimated using an equation derived from the Modification of Diet in Renal Disease study13.
Statistical Methods
For continuous variables, N, means, medians, standard deviations, minimums, and maximums were calculated. For discrete variables, numbers and percentages were calculated.
RESULTS
Of the 61 enrolled patients, 57 were found by genotyping to carry a mutation predictive of Fabry disease. All reported data pertain to these confirmed patients except where indicated otherwise. Demographic characteristics of these women are listed in Table 1.
Demographic Characteristics (n = 57)
Fabry-Specific Findings
Angiokeratomas found by physician assessment were present in 36 patients (63%). Corneal epitheliopathy by slit lamp examination was seen in 47 patients (82%). Plasma and leukocyte α-Gal A activity ranged widely, with 27 patients (47%) and 48 patients (84%), respectively, having values below the normal range. Plasma α-Gal A activity was 3.2 ± 2.25 nmol/h per mL (range, 1.49-15.90; normal range, 2.4-23.1). Leukocyte α-Gal A activity was 28.2 ± 17.85 nmol/hr per mg protein (range, 3.99-76.70; normal range, 46-126.4). Plasma Gb3 level was 6.4 ± 1.83 g/mL (range, 2.1-10.6; normal range, 5.1 ± 1.8). Twenty-four-hour urine was 331.8 ± 325.1 nmol/g creatinine per 24 h (range, 25-1492.7) (mean in 4 unconfirmed patients: 22.7 ± 13.07). There was minimal accumulation of Gb3 in the superficial dermal capillary endothelial cells; only 1 patient had a non-zero score, while 56 (98%) had scores of 0 (absence of lysosomal Gb3 inclusions in vascular endothelial cells). A small amount of storage material was found in other cell types of the skin such as smooth muscle cells and perineurium. Five of 51 patients (10%) had accumulations of Gb3 in the deep endothelial cells. For smooth muscle cells, 9 of 11 patients had accumulations of Gb3 (5 patients with mild and 4 patients with moderate accumulation). Due to the nature of the biopsy procedure, it was not unexpected that only a few patients had deep vessel smooth muscle cells available for analysis. For the perineurium, 25 of 51 patients (49%) had mild (20 patients) and moderate (5 patients) accumulations of Gb3.
Table 2 shows nonspecific Fabry-associated symptoms noted in the medical history, use of concomitant medications, or the physician assessment of Fabry symptoms. More than half the patients reported acroparesthesia and/or musculoskeletal pain. Significant gastrointestinal symptoms were also common, with slightly more than half of patients reporting recurring abdominal pain and slightly less than half reporting recurring diarrhea. Most (81%) patients considered themselves active, and most reported high or moderate exercise tolerance, and high or moderate heat tolerance.
Notable Nonspecific Fabry-Associated Findings (n = 57)
On neurologic examination, 30 patients (53%) had abnormal sensory functions. Loss of temperature sensation (especially cold, and especially in the feet and lower legs) was specifically noted in 26 patients (46%). Cranial nerve evaluations were abnormal in 16 (28%) patients. Mental status was normal in all patients.
Clinical Manifestations in Heart, Kidney, and Brain
Table 3 summarizes the results of renal assessments. Proteinuria (>150 mg/24 h) and abnormal estimated GFR (<90 mL/min per 1.73 m2) were noted in 61% (23/38) and 42% (24/57) of patients who had these assessments, respectively. Of the 23 patients with proteinuria, 9 had values >500 mg/24 h, 6 had values >1000 mg/24 h, and 2 had values >2000 mg/24 h.
Renal Function Tests
Figures 1 and 2 summarize the electrocardiogram and echocardiogram abnormalities, found in 73% (38/52) and 14% (8/57) of the patients who had these assessments, respectively.
Electrocardiogram results.
Echocardiogram results.
Of the 54 patients who had a head MRI, small (<1.5 cm) infarctions were noted in 4 patients (7%). One patient had 1 thalamic lesion, another had 2 lesions in the temporal-parietal white matter, another patient had 3 lesions in the cerebellum and thalamus, and 1 patient had 5 lesions in the thalamus and the white matter. Of the 50 patients who had an MRA, 10 patients (20%) had abnormalities (dolichoectasia or stenosis/occlusion).
Table 4 shows the distribution of abnormalities in the heart, kidney, and brain in each of the 57 patients. Overall, 52 of 57 (91%) patients had at least 1 of the following: abnormal electrocardiogram, abnormal echocardiogram, low GFR, proteinuria, abnormal MRI, or abnormal MRA.
Distribution of Abnormal Test Results in Study Population by Patient Age
Pain Data
Responses to the BPI questionnaire (Table 5) demonstrate a wide range of pain severity and pain interference. Concomitant pain medications were taken by 40 patients (70%) overall during the 30 days before the study, including 13 patients (23%) who took antiepileptic drugs prophylactically for neuropathic pain (carbamazepine, diphenylhydantoin, or gabapentin) and 8 patients (14%) who took opioid analgesics (Darvocet, Tylenol with codeine, Tylox, and Vicodin). During the 7 days that patients recorded medications in a diary, 29 patients took pain medication (51%), including 6 patients who chose to resume their antiepileptic pain medication and 4 who chose to resume their opioid analgesics.
Responses to the Brief Pain Inventory Short Form
Laboratory Findings in Unconfirmed Patients
Four additional women who had symptoms of Fabry disease and a family history of Fabry disease participated in the study, but were not found to have a Fabry genotype. These women had similar nonspecific symptoms of Fabry disease: 2 had an abnormal electrocardiogram, 3 had a low estimated GFR, and 2 reported gastrointestinal symptoms. Overall, their Fabry-specific laboratory findings, other than genotype, while not ruling out Fabry disease, tended to be less suggestive of the disease. Their mean plasma and leukocyte α-Gal A activity levels were approximately twice the values of the confirmed population. None had corneal epitheliopathy, and 2 were found to have angiokeratomas (described as "rare" for 1 patient, and mild and found only on the flank for the other patient). They all had normal urinary Gb3. In the BPI, they reported somewhat higher pain levels than the confirmed patients, and also reported less relief from pain medication and a higher level of pain interference.
DISCUSSION
In the current study we found significant manifestations of Fabry disease in this group of Fabry heterozygotes. However, since these women were selected for Fabry symptoms, our data may not represent the general population of Fabry carriers. In our study population, most women suffered from acroparesthesia, musculoskeletal pain, and abdominal pain. Almost half also reported diarrhea. When evaluated clinically, of those with data available, over 70% had electrocardiographic abnormalities, over 60% had proteinuria, and over 40% had a low estimated GFR despite normal serum creatinine. However, these values varied widely: 24-h urinary protein excretion, for example, ranged from 78 to 2907 mg, and urinary Gb3 ranged from 25 to 1493 nm/g creatinine. Pain scores on the BPI short form ranged from women who reported no pain at all to those who rated their pain at 7 on a scale of 0-10.
The current study showed no obvious benchmark of disease burden, although the finding of elevated urinary Gb3 levels in all patients confirms the findings of Cable et al5 and might be a useful diagnostic indicator for women with Fabry disease. Unlike in male patients, plasma Gb3 was not elevated, nor was there significant Gb3 deposition in capillary endothelium of the skin even in patients with renal insufficiency or a history of strokes. This finding suggests that, at least in female patients, one cannot use reduction in storage material in plasma and skin as indicators of disease burden.
The heterogeneous findings perhaps reflect varying enzyme levels in different tissues. Identification of a cardiac variant19, a renal variant9,18, and intermediate disease phenotypes8 in men suggest that, overall, Fabry disease is more heterogeneous than was previously assumed. The estimated incidence of classic Fabry disease (primarily men) is approximately 1:117,00016. About twice the number of heterozygous females carry the gene. Most families have unique mutations; to date, 272 mutations in the α-Gal A gene are recorded in the Human Gene Mutation Database23.
Five women met the entry criteria and underwent the study protocol but initial molecular DNA analysis of the exons and flanking introns did not reveal any allelic variation predictive of Fabry disease. One of these women, after subsequent analysis of her Fabry-affected son, was found to carry a large Alu-mediated deletion deleting exons 5 through 7, a mutation that would yield an inactive protein. A similar mutation was reported in a Fabry family by Kornreich et al12, although the breakpoints differ slightly, suggesting a new occurrence of the mutation by a separate mutational event. None of the other 4 women was found to have this or other Alu-mediated deletions; however, a larger genomic deletion in these individuals cannot be ruled out. This illustrates the difficulty of confirmation of diagnosis using molecular means in light of the nonspecific nature of many of the disease manifestations, such as pain and gastrointestinal disturbances. In men, whose disease symptoms tend to be more defined, the average age at diagnosis is 28 or 29 years4,16,17; in females without a family history, it is probably even higher. In a 2004 study15 of baseline data from 366 patients in Europe, the mean delay in onset of symptoms to diagnosis was 13.7 years in males and 16.3 years in females. In women, as well as in men, a low α-Gal A activity is confirmation of Fabry disease; however, as verified in our population, not all women with Fabry disease have low α-Gal A activity. Thus, there is no single sign, symptom, or laboratory finding that confirms the diagnosis in all women other than mutations in the α-Gal A gene. Although certain findings such as characteristic corneal abnormalities and angiokeratomas are strong indicators of the disease, their absence does not rule out the diagnosis in either men or women.
The results of the current study qualitatively agree with those of 4 other recent analyses of Fabry heterozygotes11,14,21,28-30. Two of these studies examined adult cohorts of Fabry carriers (patients were not selected for Fabry symptoms). In a retrospective analysis of 60 obligate heterozygotes, MacDermot et al14 found that 20 women (33%) had a history of multiple and serious manifestations, such as transient ischemic attacks and cardiovascular accidents (17 patients) and renal failure (2 patients). Whybra and colleagues28-30, who prospectively examined 20 carriers from 13 families, found 18 (90%) with acroparesthesia, 10 (50%) with nausea and diarrhea, 14 (70%) with corneal opacities ("cornea verticillata"), 12 (60%) with electrocardiographic abnormalities, 11 (55%) with reduced GFR, and 11 (55%) with angiokeratomas. Guffon et al11, in a retrospective analysis of 11 female Fabry patients, found 8 (73%) with acroparesthesia, 8 (73%) with renal dysfunction, 7 (64%) with corneal opacities, 6 (55%) with angiokeratomas, and 5 (46%) with cardiovascular abnormalities. Thus, our study describes the very significant, yet heterogeneous, burden of disease in many Fabry heterozygotes.
ACKNOWLEDGMENTS
Genotyping support for this study was conducted in collaboration with the Pharmacogenetics Laboratory of Genzyme Corporation (Dr. Robert Pomponio). Plasma Gb3 was measured by the Clinical Specialty Laboratory at Genzyme Corporation.
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