Genetic screening of Fabry patients with EcoTILLING and HRM technology

<p>Abstract</p> <p>Background</p> <p>Anderson-Fabry disease (FD) is caused by a deficit of the α-galactosidase A enzyme which leads to the accumulation of complex sphingolipids, especially globotriaosylceramide (Gb3), in all the cells of the body, causing the onset of a multi-systemic disease with poor prognosis in adulthood. In this article, we describe two alternative methods for screening the <it>GLA </it>gene which codes for the α-galactosidase A enzyme in subjects with probable FD in order to test analysis strategies which include or rely on initial pre-screening.</p> <p>Findings</p> <p>We analyzed 740 samples using EcoTILLING, comparing two mismatch-specific<ul/>endonucleases, CEL I and ENDO-1, while conducting a parallel screening of the same samples using HRM (High Resolution Melting). Afterwards, all samples were subjected to direct sequencing. Overall, we identified 12 different genetic variations: -10C>T, -12G>A, -30G>A, IVS2-76_80del5, D165H, C172Y, IVS4+16A>G, IVS4 +68 A>G, c.718_719delAA, D313Y, IVS6-22C>T, G395A. This was consistent with the high genetic heterogeneity found in FD patients and carriers. All of the mutations were detected by HRM, whereas 17% of the mutations were not found by EcoTILLING. The results obtained by EcoTILLING comparing the CEL I and ENDO-1 endonucleases were perfectly overlapping.</p> <p>Conclusion</p> <p>On the basis of its simplicity, flexibility, repeatability, and sensitivity, we believe that<ul/>HRM analysis of the <it>GLA </it>gene is a reliable presequencing screening tool. This method can be applied to any genomic feature to identify known and unknown genetic alterations, and it is ideal for conducting screening and population studies.</p

Hemochromatosis Mimicked Gaucher Disease: Role of Hyperferritinemia in Evaluation of a Clinical Case

Gaucher disease is a disorder of lysosomes caused by a functional defect of the glucocerebrosidase enzyme. The disease is mainly due to mutations in the GBA1 gene, which determines the gradual storage of glucosylceramide substrate in the patient’s macrophages. In this paper, we describe the case of a 38-year-old man who clinically presented with hyperferritinemia, thrombocytopenia, leukopenia, anemia and mild splenomegaly; a diagnosis of hemochromatosis was made 10 years earlier. Re-evaluation of the clinical case led to a suspicion of Gaucher disease, which was confirmed by enzymatic analysis, which was found to be below the normal range, and genetic evaluation, which identified compound heterozygosity N370S/RecNciI. We know that patients suffering from Gaucher disease can also have high ferritin levels. Even if the mechanism underlying the changes in iron metabolism is not yet elucidated, the chronic mild inflammatory state present in these patients probably causes the storage of ferritin in macrophages, resulting in hyperferritinemia. Therefore, in the presence of few typical signs and symptoms of the disease should raise an alarm bell in the clinicians, inducing clinical suspicion of Gaucher disease. Misdiagnosis and diagnostic delay in metabolic diseases could cause irreversible organ damage and delay the start of specific therapy for these patients

Fabry disease, a complex pathology not easy to diagnose

Fabry disease is a multisystemic lysosomal storage disorder, inherited in an X-linked manner. It is a defect of metabolism of the glycosphingolipids, due to the reduction or absence of the activity of lysosomal enzyme α-galactosidase A. This reduction of activity causes the storage of globotriaosylceramide and derivatives in the lysosomes, triggering a cascade of cellular events, mainly in vascular endothelium. These events are the responsible for the systemic clinical manifestations and the renal, cardiac and cerebrovascular complications, or a combination of them. The symptomatology can lead to the premature death of patient between the fourth or fifth decade of life. The first symptoms can occur at different ages, generally in childhood, with different severity and course. Fabry disease is suspected on the basis of clinical and anamnestic-familial data, and it is confirmed by enzymatic and genetic assays. However, Fabry disease could be a pathology more complex than previously considered, and the diagnostic tests that are currently in use could be not always sufficient to confirm the clinical diagnosis. Probably, other factors could be also involved in the onset of symptomatology. In the last years, the knowledge of the disease is considerably increased but other studies are necessary to make a prompt and reliable diagnosis. An early diagnosis of Fabry disease is essential for the beginning of the enzyme replacement therapy, which can contribute to arrest its progression and improve the quality of life of patients

Alteration of proteomic profiles in PBMC isolated from patients with Fabry disease: preliminary findings

Fabry disease (FD) is an X-linked progressive multisystem disease due to mutations in the gene encoding the lysosomal enzyme α-galactosidase A (α-GalA). The deficiency in α-GalA activity leads to an intra-lysosomal accumulation of neutral glycosphingolipids, mainly globotriaosylceramide (Gb3), in various organs and systems. Enzyme replacement therapy is available and alternative therapeutic approaches are being explored. No diagnostic test, other than sequencing of the α-galactosidase A gene, is available, no biomarker has been proven useful to screen for and predict the disease, and underlying mechanisms are still elusive. The aim of this study is to identify FD specific biomarkers and to better understand the pathophysiological changes that occur over time in FD. We compared peripheral blood mononuclear cells (PBMC) from FD patients (n = 8) with control PBMC from healthy individuals (n = 6), by two-dimensional electrophoresis (2DE) and the detected differentially expressed proteins were then subjected to matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). In FD patients we identified, among the down-regulated proteins, Calnexin, Rho GDP-dissociation inhibitor 2, Rho GDP-dissociation inhibitor 1, Chloride intracellular channel protein 1; on the other hand γ-enolase, 14-3-3 protein theta, 14-3-3 protein zeta/delta, and galectin-1 were identified as up-regulated proteins. Calnexin and Rho GDP-dissociation inhibitor-1,2 are related to protein folding, signal transduction and cell proliferation. This is the first time that γ-enolase and galectin-1 are described to be up-regulated in Fabry patients. Levels of γ-enolase increase dramatically in cardiovascular accidents and cerebral trauma, whereas galectins are regulators of acute and chronic inflammation. These findings may improve our understanding of the molecular mechanisms underlying the pathology and provide new insight and knowledge for future studies in this field

Systematic DNA Study for Fabry Disease in the End Stage Renal Disease Patients from a Southern Italy Area

Background/Aims: Fabry disease (FD) is a lysosomal storage disorder characterized by pervasive renal involvement. However, this disease is underdiagnosed in patient with chronic kidney disease (CKD), including those with end stage renal disease (ESRD), so their investigation represents an unexploited opportunity for early diagnosis of the disease and for its identification in relatives of affected patients. Methods: We investigated Fabry disease in a clinical and biological database including ESRD patients of unknown cause in a geographical area with 2 million residents. The study was based on state of art GLA gene sequencing and was extended to relatives of affected ESRD patients. Results: Among ESRD patients qualified for enrollment into this study, a previously undiagnosed young man harboring the mutation p.I91T was identified. The study of the proband's family led to the identification of 8 additional cases. In another ESRD male patient, we identified the functional polymorphism p.D313Y. Furthermore, in 55 ESRD patients (24.2%) we found intronic polymorphisms of uncertain functional relevance in the non-coding regions of the GLA gene. Conclusion: A comprehensive survey of ESRD patients in a geographical area of 2 million residents identified one undiagnosed case of Fabry disease and led to the identification of 8 additional cases among his relatives. Screening protocols starting from the dialysis population and upstream extended to families of affected individuals may be an effective strategy to maximize the early identification of subjects with Fabry disease

Gaucher Disease or Acid Sphingomyelinase Deficiency? The Importance of Differential Diagnosis

Background: Gaucher disease is a lysosomal storage disorder caused by functional glucocerebrosidase enzyme deficiency. Hepatosplenomegaly and hematological complications are found in both Gaucher disease and Acid Sphingomyelinase Deficiency, which is caused by acid sphingomyelinase dysfunction. The possible overlap in clinical presentation can cause diagnostic errors in differential diagnosis. For this reason, in patients with an initial clinical suspicion of Gaucher disease, we aimed to carry out a parallel screening of acid sphingomyelinase and glucocerebrosidase. Methods: Peripheral blood samples of 627 patients were collected, and enzymatic activity analysis was performed on both glucocerebrosidase and acid sphingomyelinase. The specific gene was studied in samples with null or reduced enzymatic activity. Specific molecular biomarkers helped to achieve the correct diagnosis. Results: In 98.7% of patients, normal values of glucocerebrosidase activity excluded Gaucher disease. In 8 of 627 patients (1.3%), the glucocerebrosidase enzymatic activity assay was below the normal range, so genetic GBA1 analysis confirmed the enzymatic defect. Three patients (0.5%) had normal glucocerebrosidase activity, so they were not affected by Gaucher disease, and showed decreased acid sphingomyelinase activity. SMPD1 gene mutations responsible for Acid Sphingomyelinase Deficiency were found. The levels of specific biomarkers found in these patients further strengthened the genetic data. Conclusions: Our results suggest that in the presence of typical signs and symptoms of Gaucher disease, Acid Sphingomyelinase Deficiency should be considered. For this reason, the presence of hepatosplenomegaly, thrombocytopenia, leukocytopenia, and anemia should alert clinicians to analyze both enzymes by a combined screening. Today, enzyme replacement therapy is available for the treatment of both pathologies; therefore, prompt diagnosis is essential for patients to start accurate treatment and to avoid diagnostic delay