Research On Diagnostic Methods Of Amyloidosis With Familial Amyloidotic Polyneuropathy Patients

Aims: 1.Transthyretin (TTR), a visceral protein functioning as transporter of thyroxine and vitamin A, mainly found in circulation and cerebrospinal fluid, is amyloid precursor protein related to familial amyloidotic polyneuropathy (FAP) and senile systemic amyloidosis(SSA). It was evidenced that TTR amyloid formation can be enhanced by gene mutation or (and) environmental factors. For the pathogenesis and treatment differences between FAP and SSA amyloidosis, it is quite important to diagnose these two kinds of amyloidosis at the molecular level.However ,there are no ideal molecular diagnostic methods and techniques of TTR-related amyloidosis.To explore a new diagnostic method of TTR-related amyloidosis, can be useful for the clinical diagnosis and treatment of TTR-related amyloidosis. 2.To explore the amyloid formation mechanism in FAP patients by analyzing the TTR amyloidosis deposition extent and morphological characteristics in the heart and digestive tract tissues.Methods: 1 .Tissues from patients with FAP were collected for sections and freezing.Sections was stained by Congo red (CR) and observed under polarized microscope,for which CR and birefringence positive, immunohistochemistry was used for subtyping amyloid proteins.A fluorescent probe was designed to specific bind to the amyloidosis. A rapid and specific diagnosis method of amyloidosis was established.The TTR-related genes were amplification to analyze the gene mutation type. Matrix assisted laser desorption ionization time-of-flight mass spectrometry ( MALDI-TOF/MS) was used to determine the gene mutation type of TTR. 2. Amyloid deposition extent and morphological characteristics were analyzed with histological morphology analysis.Results: 1.Amyloid was found in kidney, heart, small intestine and cerebellar tissues of FAP patients, as confirmed by CR and apple green birefringence under polarized light microscopy. DNA was extracted from blood cells of FAP patients, and the genotype was confirmed as ATTR Val30Met after PCR amplification and DNA sequencing of PCR results. The serum TTR mass characteristics of the same patients was analyzed using MALDI-TOF/MS to develop the MALDI-TOF/MS method standard for the determination of TTR. The amyloid precursor protein was demonstrated to be TTR by immunohistochemistry. There was affinity between the fluorescent probe and amyloidosis fibers, while no obvious affinity between the fluorescent probe and the serum TTR protein. Further observation of the affinity of the labeled probe and tissue deposition of amyloidosis showed that the fluorescent probe stains had the same result with CR stains. 2. Amyloid was found in small blood vessels and myocardial interstitial in heart tissues, as confirmed by Congo red stain and apple green birefringence under polarized light microscopy.Conclusion: 1.For the first time we reported the MALDI-TOF/MS method to directly determine the serum TTR protein mutation types.And it can be used for the clinical detection of TTR mutation and chemical modification. We designed and synthesized a fluorescent probe that can combine with amyloidosis fibers.The fluorescence method for rapid diagnosis of amyloidosis is suitable for clinical diagnosis amyloidosis,especially in clinical surgery. 2. Amyloid deposition is significant in myocardial interstitial and loose connective tissues of FAP ATTR Val30Met patients, suggesting that factors such as carrier transportation and chemical modification may play important roles in TTR amyloidosis.