The Anderson-Fabry disease is an inherited disorder of glycosphingolipids’ metabolism caused by a deficiency of the lysosomal enzyme alfa-galactosidase A, whose transmission is X-linked recessive. The definitive diagnosis is performed with the demonstration of a deficiency of alfa-galactosidase A activity in plasma, leukocytes, or in the biopsy specimens, and confirmed by genetic investigation of specific mutations. In 2004, at the Policlinico San Matteo in Pavia started a national multicenter prospective project aimed at diagnosis of Fabry disease, involving a high number of probands to whome the enzymatic test was performed in plasma and leukocytes samples, as well as the genetic test. My thesis work took place over a 6 months period during which I have applied an analytical procedure already established and previously validated for the determination of residual enzymatic activity of alfa-galactosidase A in plasma samples of and leukocytes as well, carried out by a fluorimetric method. From the resulting residual enzymatic activity obtained in plasma and leukocytes in females and males both mutated and wt for Fabry Disease, a Receiver Operating Characteristic curve (ROC Curve) was obtained in order to identify the optimal threshold and to determine the best cut- off, allowing to discriminate with the best specificity and sensitivity, true positives and true negatives within the population. Although the symptoms of Fabry Disease usually appear during childhood, they may not be recognized until adult age, when an organ damage has already arose. Is therefore essential to make an early diagnosis, as this is a progressive and maybe a life-threatening disease. In males the definitive diagnosis can be given by the demonstration of a lack of activity of alfa-Gal in plasma/leukocyte or tissue biopsies. It should be noted that, since the female carriers can present values of the activity even normal, only the mutation or linkage analysis can definitively identify the heterozygous female ones. According to the scientific literature, the determination of the enzyme activity of alfa-Gal carried out into leukocytes is recognized as the method of choice (gold standard). In fact, the use of plasma matrix alone may give false negative results, especially in females carriers. For these reasons it is important to perform the assay in at least two different matrices, in order to unmask the pseudodeficiency of activities that can lead to misdiagnosis. In our laboratory we are always performing the enzymatic test, where possible, both into leukocytes and in the plasma matrices.
La Malattia di Anderson-Fabry è una patologia ereditaria del metabolismo dei glicosfingolipidi, causata da un deficit dell’enzima lisosomiale alfa-galattosidasi A, la cui trasmissione recessiva è legata al cromosoma X. La diagnosi definitiva viene posta con la dimostrazione di un deficit di attività di alfa-galattosidasi A nel plasma, nei leucociti o nel campione bioptico, e successiva conferma genetica mediante ricerca della mutazione specifica. Metodi Nel 2004 presso il Policlinico San Matteo di Pavia è iniziato un progetto nazionale multicentrico prospettico finalizzato alla diagnosi della Malattia di Fabry che ha coinvolto un elevato numero di probandi sui quali è stato effettato il test diagnostico per l’analisi della attività enzimatica residua dell’enzima alfa-galattosidasi A su campioni di plasma e di leucociti, nonché la sequenziazione del gene GLA. La mia attività di tesi si è svolta in un periodo di 6 mesi durante i quali ho applicato una procedura analitica già consolidata e precedentemente validata per il dosaggio della attività enzimatica residua di alfa-galattosidasi A su campioni di plasma e di leucociti, effettuata mediante un metodo fluorimetrico. Dopo aver effettuato la valutazione del valore di attività enzimatica nel plasma e nei leucociti in femmine e maschi mutati e wt, è stata ottenuta una curva ROC (Receiver Operating Characteristic) per identificare la soglia ottimale per il test plasmatico e leucocitario e stabilire il cut-off ottimale per entrambi che permetta di discriminare con la migliore specificità e sensibilità i veri positivi dai veri negativi. Sebbene i sintomi della malattia compaiano generalmente durante l'infanzia, possono non essere riconosciuti fino all'età adulta, quando si è già verificato un danno d’organo. E’ quindi fondamentale effettuare una diagnosi precoce, dal momento che la malattia è progressiva e può essere pericolosa per la vita. Quasi tutti i maschi malati possono essere facilmente diagnosticati con analisi nel plasma per la quasi assenza di attività di alfa-Gal nella matrice plasmatica; è da notare che, poiché le femmine portatrici possono presentare valori normali della attività dell’enzima, la mutazione o l’analisi del linkage possono identificare definitivamente tali femmine portatrici eterozigote. La determinazione della attività enzimatica effettuata sui leucociti è tuttavia riconosciuta come metodo di scelta (gold standard). L’uso della sola matrice plasmatica può dare infatti risultati falsi negativi, in particolare modo nelle femmine. Per questi motivi è importante effettuare il dosaggio in almeno due diverse matrici, in modo da smascherare le pseudodeficienze di attività che possono portare a diagnosi errate. In questo laboratorio vengono quindi effettuate sempre, ove possibile, le determinazioni sia sulla matrice dei leucociti che nel plasma.
Malattia di Anderson-Fabry: screening di popolazione mediante l'analisi dell'enzima alfa-galattosidasi A
ZAVATTARO, MARTA
2014/2015
Abstract
The Anderson-Fabry disease is an inherited disorder of glycosphingolipids’ metabolism caused by a deficiency of the lysosomal enzyme alfa-galactosidase A, whose transmission is X-linked recessive. The definitive diagnosis is performed with the demonstration of a deficiency of alfa-galactosidase A activity in plasma, leukocytes, or in the biopsy specimens, and confirmed by genetic investigation of specific mutations. In 2004, at the Policlinico San Matteo in Pavia started a national multicenter prospective project aimed at diagnosis of Fabry disease, involving a high number of probands to whome the enzymatic test was performed in plasma and leukocytes samples, as well as the genetic test. My thesis work took place over a 6 months period during which I have applied an analytical procedure already established and previously validated for the determination of residual enzymatic activity of alfa-galactosidase A in plasma samples of and leukocytes as well, carried out by a fluorimetric method. From the resulting residual enzymatic activity obtained in plasma and leukocytes in females and males both mutated and wt for Fabry Disease, a Receiver Operating Characteristic curve (ROC Curve) was obtained in order to identify the optimal threshold and to determine the best cut- off, allowing to discriminate with the best specificity and sensitivity, true positives and true negatives within the population. Although the symptoms of Fabry Disease usually appear during childhood, they may not be recognized until adult age, when an organ damage has already arose. Is therefore essential to make an early diagnosis, as this is a progressive and maybe a life-threatening disease. In males the definitive diagnosis can be given by the demonstration of a lack of activity of alfa-Gal in plasma/leukocyte or tissue biopsies. It should be noted that, since the female carriers can present values of the activity even normal, only the mutation or linkage analysis can definitively identify the heterozygous female ones. According to the scientific literature, the determination of the enzyme activity of alfa-Gal carried out into leukocytes is recognized as the method of choice (gold standard). In fact, the use of plasma matrix alone may give false negative results, especially in females carriers. For these reasons it is important to perform the assay in at least two different matrices, in order to unmask the pseudodeficiency of activities that can lead to misdiagnosis. In our laboratory we are always performing the enzymatic test, where possible, both into leukocytes and in the plasma matrices.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/23994