We analyzed 11 consecutive unrelated cases of polyneuropathy due to transthyretin amyloidosis. Direct sequencing of the promoter region, exons, and splice junctions revealed that each patient was heterozygous for a mutation: six patients had valine 30 substituted by methionine (V30----M; Portuguese-Japanese type), one had threonine 60 substituted by alanine (T60----A; Appalachian type), and two had serine 77 substituted by tyrosine (S77----Y; Illinois type). In addition, two patients had previously undescribed mutation: phenylalanine 33 substituted by leucine (F33----L) and phenylalanine 64 substituted by leucine (F64----L). From present information, the probands of these novel mutations do not exhibit any pathology that clearly distinguishes them from individuals with the other mutations. The mutations extend the range of mutations associated with amyloidotic polyneuropathy. In our 11 patients, the different mutations did not seem to correlate with distinct clinical phenotypes. We developed PASA assays (PCR amplification of specific alleles) for each of the five mutations. PASA can be used by any diagnostic laboratory that can perform PCR to rapidly detect any of the known mutations. The minority of samples with an undescribed mutation can be sent to a specialty laboratory for delineation of the mutation by direct genomic sequencing. The presently described combination of methods may have widespread utility in the diagnosis of genetic disease.
Human liver guanase was purified and a specific antibody against it was raised in rabbits. The antiserum formed a single precipitin line with human liver extract, and also completely inhibited the activity of the liver enzyme. An immunoblotting study showed that the antibody bound specifically to one band of protein with guanase activity and not to other proteins. Therefore, we concluded that this antiserum against the liver enzyme was suitable for use in immunohistochemical demonstration of guanase. In tissue sections, the immunohistochemical reaction with this antibody was positive in the same locations as the histochemical guanase reaction with DAB (3,3'-diaminobenzidine tetrahydrochloride).
: Guanase is known as an enzyme released from the liver. Recently, cloning and sequencing of the guanase gene were reported. In addition, almost simultaneously, it was reported that an unknown protein that binds to neuronal and endocrine lethal(1)-discs large (NE-dlg), one of the membrane-associated guanylate kinase homologues (MAGUK) family proteins involved in synaptic connection between neurons, was cloned and named nedasin (NE-dlg associated protein), whose sequence was almost identical to that of guanase. We immunostained fresh frozen sections of surgically removed human liver, kidney, and small intestine with anti-nedasin antibody, and simultaneously performed histochemical staining for guanase for comparison. Histochemically, guanase activity was observed in the cytoplasm of hepatocytes and biliary epithelium on the liver, in the mucosal epithelium on the small intestine, and in the proximal tubule on the kidney. Immunohistochemically, a brown discoloration due to DAB oxidation was seen in the cytoplasm of hepatocytes and biliary epithelium on the liver, in the proximal tubule but in the distal tubule a little on the kidney, in the mucosal epithelium on the small intestine. The stained region of the liver and the small intestine were different from that of the kidney. The different staining properties dependent on the organs were considered to be due to different isozymes. The physiological significance of guanase may vary with the isozymes, further studies are considered necessary.
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