CD77 levels over enzyme replacement treatment in Fabry Disease Family (V269M) / Níveis de CD77 ao longo do tratamento de reposição enzimática em família com Doença de Fabry (V269M)

ABSTRACT Introduction: Fabry disease (FD) is a disorder caused by mutations in the gene encoding for lysosomal enzyme α-galactosidase A (α-GAL). Reduced α-GAL activity leads to progressive accumulation of globotriaosylceramide (Gb3), also known as CD77. The recent report of increased expression of CD77 in blood cells of patients with FD indicated that this molecule can be used as a potential marker for monitoring enzyme replacement therapy (ERT). Objective: The purpose of this study was to evaluate the CD77 levels throughout ERT in FD patients (V269M mutation). Methods: We evaluated the fluctuations in PBMC (peripheral blood mononuclear cell) membrane CD77 expression in FD patients undergoing ERT and correlated these levels with those observed in different cell types. Results: A greater CD77 expression was found in phagocytes of patients compared to controls at baseline. Interestingly, the variability in CD77 levels is larger in patients at baseline (340 - 1619 MIF) and after 12 months of ERT (240 - 530 MIF) compared with the control group (131 - 331 MFI). Furthermore, by analyzing the levels of CD77 in phagocytes from patients throughout ERT, we found a constant decrease in CD77 levels. Conclusion: The increased CD77 levels in the phagocytes of Fabry carriers together with the decrease in CD77 levels throughout ERT suggest that measuring CD77 levels in phagocytes is a promising tool for monitoring the response to ERT in FD.

RESUMO Introdução: A doença de Fabry (DF) é um distúrbio causado por mutações no gene que codifica a enzima lisossômica α-galactosidase A (α-GAL). A redução da atividade de α-GAL leva ao acúmulo progressivo de globotriaosilceramida (Gb3), também conhecida como CD77. O recente relato de aumento da expressão de CD77 em células sanguíneas de pacientes com DF indicou que essa molécula pode ser utilizada como um potencial marcador para o monitoramento da terapia de reposição enzimática (TRE). Objetivo: O objetivo deste estudo foi avaliar os níveis de CD77 ao longo da TRE em pacientes com DF (mutação V269M). Métodos: Foram avaliadas as flutuações na expressão de CD77 nas membranas das CMSP (células mononucleares do sangue periférico) em pacientes com DF submetidos à TRE e correlacionados com aqueles observados em diferentes tipos de células. Resultados: Uma maior expressão de CD77 foi encontrada em fagócitos de pacientes em comparação aos controles no início do estudo. Curiosamente, a variabilidade nos níveis de CD77 é maior em pacientes no início do estudo (340 - 1619 MIF) e após 12 meses de TRE (240 - 530 MIF) em comparação com o grupo controle (131 - 331 MFI). Além disso, analisando os níveis de CD77 em fagócitos de pacientes ao longo da TRE, encontramos uma diminuição constante nos níveis de CD77. Conclusão: O aumento nos níveis de CD77 nos fagócitos de portadores de Fabry, juntamente com a diminuição nos níveis de CD77 ao longo da TRE, sugerem que medir os níveis de CD77 nos fagócitos é uma ferramenta promissora para monitorar a resposta à TRE na DF.

Analysis of Lyso-Globotriaosylsphingosine in Dried Blood Spots

Recently, lyso-globotriaosylsphingosine (lyso-Gb3) was found to be elevated in plasma of treatment naive male patients and some female patients with Fabry Disease (FD). This study tested whether lyso-Gb3 could be analyzed in dried blood spots (DBS) from filter cards and whether concentrations are elevated in newborn infants with FD. Lyso-Gb3 concentrations were analyzed in DBS following extraction using a novel HPLC-mass spectrometry (MS)/MS method. Lyso-Gb3 levels in DBS were above the lower limit of quantitation (0.28 ng/mL) in 5/17 newborn FD infants (16 males; range: 1.02-8.81 ng/mL), but in none of the newborn controls, in all 13 patients (4 males) with classic FD (range: 2.06-54.1 ng/mL), in 125/159 Taiwanese individuals with symptomatic or asymptomatic FD who carry the late onset alpha-galactosidase A (GLA) mutation c.936+919G>A (IVS4+919G>A) (3.75+/-0.69 ng/mL; range: 0.418-3.97 ng/mL) and in 20/29 healthy controls (0.77+/-0.24 ng/mL; range: 0.507-1.4 ng/mL). The HPLC-MS/MS method for analysis of lyso-Gb3 is robust and yields reproducible results in DBS in patients with FD. However, concentrations of lyso-Gb3 were below the limit of quantitation in most newborn infants with FD rendering this approach not suitable for newborn screening. In addition, most females with the late onset mutation have undetectable lyso-Gb3 concentrations.

Serum Globotriaosylceramide Assay as a Screening Test for Fabry Disease in Patients with ESRD on Maintenance Dialysis in Korea

BACKGROUND/AIMS: Fabry disease is an X-linked recessive and progressive disease caused by alpha-galactosidase A (alpha-GaL A) deficiency. We sought to assess the prevalence of unrecognized Fabry disease in dialysis-dependent patients and the efficacy of serum globotriaosylceramide (GL3) screening. METHODS: A total of 480 patients of 1,230 patients among 17 clinics were enrolled. Serum GL3 levels were measured by tandem mass spectrometry. Additionally, we studied the association between increased GL3 levels and cardiovascular disease, cerebrovascular disease, or left ventricular hypertrophy. RESULTS: Twenty-nine patients had elevated serum GL3 levels. The alpha-GaL A activity was determined for the 26 patients with high GL3 levels. The mean alpha-GaL A activity was 64.6 nmol/hr/mg (reference range, 45 to 85), and no patient was identified with decreased alpha-GaL A activity. Among the group with high GL3 levels, 15 women had a alpha-GaL A genetics analysis. No point mutations were discovered among the women with high GL3 levels. No correlation was observed between serum GL3 levels and alpha-GaL A activity; the Pearson correlation coefficient was 0.01352 (p = 0.9478). No significant correlation was observed between increased GL3 levels and the frequency of cardiovascular disease or cerebrovascular disease. CONCLUSIONS: Fabry disease is very rare disease in patients with end-stage renal disease. Serum GL3 measurements as a screening method for Fabry disease showed a high false-positive rate. Thus, serum GL3 levels determined by tandem mass spectrometry may not be useful as a screening method for Fabry disease in patients with end stage renal disease.

Short-Term Efficacy of Enzyme Replacement Therapy in Korean Patients with Fabry Disease

Fabrazyme has been widely used for treatment of Fabry disease since its approval by the U.S. Food and Drug Administration in 2003. This study was undertaken to assess the short-term efficacy and safety of enzyme replacement therapy (ERT) for Fabry disease in Korea. Eight male patients and three female symptomatic carriers aged 13 to 48 yr were included. Fabrazyme was administered by intravenous infusion at a dose of 1 mg/kg every 2 weeks. Plasma and urine globotriaosylceramide (GL-3) levels, serum creatinine, creatinine clearance, and 24-hr urine protein levels were measured every 3 months. Kidney biopsies, ophthalmologic exams, and pure tone audiometry were performed before and 1 yr after ERT. Kidney function, including serum creatinine, creatinine clearance, and the 24-hr urine protein level, remained stable during ERT. Plasma and urine GL-3 levels were reduced within 3 to 6 months of ERT initiation. Microvascular endothelial deposits of GL-3 were decreased from renal biopsy specimens after 1 yr of treatment. The severity of sensorineural hearing loss and tinnitus did not improve after ERT. ERT is safe and effective in stabilizing renal function and clearing microvascular endothelial GL-3 from kidney biopsy specimen in Korean patients with Fabry disease.

Clinical and molecular characterization of an extended family with Fabry disease.

OBJECTIVE: To characterize clinical manifestations, biochemical changes, mutation of alpha-Galactosidase (alpha-Gal A) gene A (GLA), and functional capability of mutant protein. MATERIAL AND METHOD: Seventeen subjects from a family with a newly diagnosed patient with Fabry disease were enrolled in the present study. In each individual, clinical history, physical examination, leukocyte enzyme activity of alpha-Gal A, and mutation analysis were performed. Those with a mutation were further investigated by ophthalmological and audiological evaluations, electrocardiography, echocardiogram, urinalysis, and blood tests to determine renal insufficiency. Expression study of the mutant protein was performed using a Pichia pastoris expression system. RESULTS: Four affected males and five symptomatic female carriers were identified. Clinical manifestations included severe neuropathic pain, acroparesthesia, hypo-/hyper-hidrosis, frequent syncope, ischemic stroke, cardiac hypertrophy, corneal dystrophy and cart-wheel cataract, high frequency sensorineural hearing loss, periorbital edema and subcutaneous edema over hands and interphalangeal joints. None had angiokeratoma or renal symptoms. The authors identified a novel mutation, p.L106R, in the GLA gene. Recombinant expression of the mutant protein gave little or no enzyme activity compared to the normal protein. CONCLUSION: There were intrafamilial clinical variabilities, but consistent findings of the absence of angiokeratoma and renal symptoms, which could represent a unique feature of this particular mutation.