Shinichi Ikegawa

Systemic amyloidosis in transgenic mice carrying the human mutant transthyretin (Met 30) gene

To analyze the pathologic processes of amyloid deposition in type I familial amyloidotic polyneuropathy (FAP), mice were made transgenic by introducing the human mutant transthyretin (TTR) gene(MT-hMet 30). An inbred strain of mouse, C57 BL/6, was chosen. Transgenic mice were killed using ether anesthesia at 3-mo intervals up to 24 mo after birth. In these transgenic mice, amyloid deposition started in the gastrointestinal tract, cardiovascular system, and kidneys and extended to various other organs and tissues with advancing age. The pattern of amyloid deposition was similar to that observed in human autopsy cases of FAP, except for its absence in the choroid plexus and in the peripheral and autonomic nervous systems.We extracted the amyloid fibrils from kidneys of these mice with a human mutant TTR gene and analyzed them immunochemically and electronmicroscopically. Deposited amyloid was shown to be composed of human mutant TTR and mouse serum amyloid P component. Amyloid fibril from transgenic mice was morphologically and immunohistochemically similar to that of human FAP.The most striking pathologic feature of the transgenic mice was the absence of amyloid deposition in the peripheral and autonomic nervous tissues. Thus, other intrinsic factors may be involved in amyloid deposition in the nervous tissues of human FAP.

A novel transthyretin mutation associated with familial amyloidotic polyneuropathy.

We characterized the mutation associated with familial amyloidotic polyneuropathy in a Japanese patient. Sequence analysis of polymerase chain reaction-amplified exons of the transthyretin gene revealed a novel point mutation resulting in a substitution of arginine for glycine at position 47. The mutation was confirmed using allele-specific olgonucleotide hybridization procedures. This most likely represents a de novo mutation since neither parent carries the mutant allele.

Disturbed metabolism of glucose and related hormones in familial amyloidotic polyneuropathy: hypersensitivities of the autonomic nervous system and therapeutic prevention.

Regulation of glucose metabolism was evaluated by oral glucose tolerance test (OGTT) in patients with familial amyloidotic polyneuropathy (FAP). Upon oral administration of a loading dose of glucose, plasma levels of glucose, insulin and glucagon changed abnormally in all FAP patients tested. Although plasma levels of glucose and insulin in the fasted patients were within normal ranges, 33% of FAP patients showed hypoglycemia after transient hyperinsulinemia during the examination. Furthermore, another three patients showed transient hypoglycemia during their daily life. Thus, perturbed glucose metabolism should be taken into account for treating patients with FAP. The salivary glands as well as the lacrimal glands showed transient hypersecretion after chewing a gum. Histochemical analysis at autopsy revealed significant amyloid deposition in the stroma, nerves and vessels of the pancreas, but not in Langerhans islets. Similar appearance was recognized in the salivary glands. These results suggest that denervation supersensitivity might occur not only in the exocrine glands but also in the endocrine gland.

Identification of carriers of mutant prealbumin gene associated with familial amyloidotic polyneuropathy type I by Southern blot procedures: study of six pedigrees in the Arao district of Japan

SummaryFifty-six Japanese individuals from six pedigrees with familial amyloidotic polyneuropathy (FAP), together with 2 individuals with symptomatic FAP from an unknown pedigree were analyzed, using the Southern blot procedures for the prealbumin gene structure. A human prealbumin cDNA was used as the probe. Altogether, these individuals included 20 with symptomatic FAP, 30 who were asymptomatic, and 8 disease-free spouses. Twenty individuals with symptomatic FAP were all heterozygous for the prealbumin genes, carrying one normal and one mutant gene. We confirmed the direct linkage between the mutant prealbumin gene and the Japanese type of FAP. Moreover, 10 of the 30 asymptomatic individuals from pedigrees with FAP were also heterozygous for the prealbumin gene. The number of asymptomatic individuals with the mutant prealbumin gene showed age-related decreases, and none was over 40 years. A linkage between the mutant prealbumin gene and serum levels of the prealbumin variant was also evident.

Role of variant prealbumin in the pathogenesis of familial amyloidotic polyneuropathy: Fate of normal and variant prealbumin in the circulation

According to recent studies on protein chemistry and genetic engineering, replacement of the Val30 residue of prealbumin by methionine is believed to play a critical role in the formation of amyloid deposit and the pathogenesis of familial amyloidotic polyneuropathy (FAP). However, only limited information is available concerning the behavior of prealbumin in the circulation. To obtain the molecular insight into the mechanism of amyloid deposition, it is indispensable to know the fates of normal and variant prealbumin in vivo. Thus, the fates of prealbumin samples from normal and FAP patients were studied in normal rats as well as in animals that were challenged with acute inflammation induced by turpentine. The effect of in vitro photooxidation of prealbumin samples on their behavior was also examined in vivo. Kinetic analysis revealed no appreciable difference between prealbumin samples from normal and FAP patients. These results suggest that factors other than the rate of transfer of the variant form prealbumin from plasma to an extravascular compartment may play a critical role in the pathogenesis of amyloid deposition in FAP patients.

Quantitative detection of a variant prealbumin associated with type 1 familial amyloidotic polyneuropathy (japanese type) by high performance liquid chromatography

A simple and quantitative method for detecting the variant prealbumin associated with familial amyloidotic polyneuropathy has been developed. This method is based on (1) a rapid and simple high performance liquid chromatographic method for the purification of prealbumin, using an immunoadsorbent-affinity column with bound monospecific prealbumin antibody, (2) the presence of an extra methionine in the variant prealbumin at position 30, detected by cyanogen bromide cleavage, and (3) sensitive and quantitative detection of cleaved peptides by reversed phase high performance liquid chromatography. This non-radioisotopic method gives quantitatively reliable results on serum samples as small as 0.5 ml. This method is not only useful for the detection of patients and carriers of familial amyloidotic polyneuropathy, but also for determination of the ratio of normal to variant prealbumin in the serum samples.

Evaluation of Serum Variant Prealbumin Levels, and its Behavior after Plasma Exchange for Familial Amyloidotic Polyneuropathy (Type 1)

Type 1 familial amyloidotic polyneuropathy (FAP) is an autosomal dominant systemic amyloidosis, characterized by extracellular amyloid deposition and sensory dominant polyneuropathy accompanied by autonomic dysfunction.1 2. Biochemical research disclosed that the amyloid fibril protein in type 1 FAP consists of a variant prealbumin (PA) (transthyretin), due to a single amino acid substitution, a methionine for valine at position 30.3 5 Recently, FAP and its carrier state were identified by the detection of the variant PA in serum.6 9

Reevaluation of 134 Patients with Familial Amyloidotic Polyneuropathy (FAP) in Japan, Kumamoto Focus

Clinical data on FAP in Kumamoto area, Japan from 1967 to 1990, were reevaluated in March 1990. One hundred, thirty four patients in 8 pedigrees, male 70 and female 64 were studied. The initial symptoms occured at 18-69 years of age (mean 34.2) with sensory disturbance at lower extremities (50%), or constipation / diarrhea (33.9%). The duration of illness was 5-19 years (mean 12.5 years). Female patients had a tendency of late onset (36.9 years old) and a long duration of illness (12.5 years). Main clinical symptoms were polyneuropathy, autonomic dysfunction and general manifestations such as edema, arrhythmia and visual disturbances. The diagnosis was made by biopsy, DNA and serum diagnosis. Owing to the recent diagnostic advances, seven late onset cases who had initial symptoms later than 45 years old have been found. Symptomatic treatment such as L-DOPS, DMSO, pace-maker implantation, opthalmological operations, hemodialysis, and other, were not only useful to eliminate subjective symptoms of the disease but also extended the survival from 8.8 years to 9.4 years, compared with those observed in an epidemiological study done in 1982 [1].

Gastrointestinal Malabsorption in Familial Amyloidotic Polyneuropathy (FAP): Ursodeoxycolic Acid Absorption Test

We applied to use ursodeoxycolic acid (UDCA), which is often used for the evaluation of liver dysfunction, as the useful tool evaluating the grades of gastrointestinal malabsorption in FAP patients. In most of FAP patients, serum levels of total bile acid were not sufficiently elevated after administration of UDCA. Interestingly, oral administration of L-threo-3,4-dihydroxyphenyl-seline (L-threoDOPS), the artificial precursor of norepinephrine, improved the malabsorption of UDCA when orally administered in FAP patients with mild and moderate diarrhea. This may be because L-threoDOPS suppress the hyperperstarsis of the gastrointestinal tract in FAP patients by elevating serum norepinephrine levels. Moreover, nasal application of L-threoDOPS, the mixture of L-threoDOPS and others, elevated serum levels of total bile acid in FAP patients with severe diarrhea. Procaterol hydrochloride, s2-Stimulantr also showed significant anti-diarrhea effect in mild and moderate diarrhea. These results suggest that malabsorption and diarrhea found in FAP patients may be improved by oral and nasal administration of adrenergic drugs.

Detection of Autonomic Dysfunctions in Patients with Familial Amyloidotic Polyneuropathy (FAP) by Laser Doppler Method

Laser Doppler flowmetry (LDF) examination was conducted in 11 FAP patients, 3 asymptomatic carriers of FAP and 29 normal controls. The vasoconstrictive responses induced by deep inspiration in the 11 FAP patients were markedly depressed, compared with those in healthy volunteers. In the 7 patients advanced to the stages 2 (moderate) to 4 (terminal), the responses were not elicited at all. To our interest, the vasoconstrictive responses following deep inspiration were also depressed in two of the 3 asymptomatic carriers of FAP, who had normal sensory nerve conduction velocity. Some patients with other diseases, such as Shy-Drager disease and pandysautonomia, also failed to exhibit the decrease of blood flow for various stimulations. Thus, these data suggest that the autonomic nervous system, especially the sympathetic vasomotor nerve regulating peripheral circulation, might be affected prior to the somatic polyneuropathy in FAP patients.