Shigehiro Yi

Familial amyloidotic polyneuropathy type 1 in Kumamoto, Japan: A clinicopathologic, histochemical, immunohistochemical, and ultrastructural study

Seventeen autopsy and five biopsy cases of familial amyloidotic polyneuropathy were examined clinicopathologically, histochemically, immunohistochemically, and ultrastructurally. In the autopsy cases, amyloid deposits were predominant in the peripheral nerve tissues, autonomic nervous system, choroid plexus, cardiovascular system, and kidneys. Amyloid involvements in the anterior and posterior roots of the spinal cord, spinal ganglia, thyroid, and gastrointestinal tract were also frequent. In the cardiac conduction system, amyloid deposition was prominent in the sinoatrial node and in limbs of the intraventricular bundle. In the sural nerve biopsy, besides amyloid deposits, degenerative changes of nerve fibers and Schwann cells were detected ultrastructurally, and the morphometric analysis showed a marked reduction in the number of myelinated fibers which correlated with the clinical stage. Amyloid deposits were resistant to pretreatment with potassium permanganate in Congo red staining, and transthyretin was confirmed immunohistochemically as a major component of amyloid deposits, along with the presence of serum amyloid P-component. Besides the amyloid deposits, transthyretin was proven in the liver cells, epithelial cells of the choroid plexus, and pancreatic islet A cells, suggesting that the transthyretin produced by these cells is secreted, transferred into tissues, and deposited in situ as the major component of amyloid in this disorder.

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.

Comparison of amyloid deposition in two lines of transgenic mouse that model familial amyloidotic polyneuropathy, type I.

We previously produced a transgenic mouse line designated MT-hMet30 by introducing the human mutant transthyretin (TTR) gene carrying the mouse metallothionein promoter, and showed that the presence of human variant TTR is sufficient for amyloid deposition in various tissues of these transgenic mice. However, the expression pattern of human mutant transthyretin gene in the mouse was different from that in man. To analyse pathologic processes, it is essential to establish a transgenic mouse line in which the developmental and tissue- specific expression of the human mutant TTR gene is the same as in man. Thus, we produced two additional transgenic mouse lines carrying the human mutant TTR gene containing either 0.6 kb (0.6- hMet30) or 6.0 kb (6.0-hMet30) of the upstream region. The expression levels of 6.0-hMet30 gene in the liver and serum were the same as in man and about 10 times higher than those of 0.6- hMet30 gene. In both lines amyloid deposition was observed in similar tissues to human patients except for the peripheral and autonomic nervous tissues. The amyloid deposition started earlier and was more extensive in 6.0-hMet30 than 0.6-hMet30 mice, suggesting that the serum levels of human mutant TTR are correlated with the occurrence and degree of amyloid deposition, to some extent. Neither amyloid deposition nor degenerative changes were observed in the peripheral and autonomic nervous systems despite the transgene expression in the choroid plexus of the 6.0-hMet30 mice. In the 6.0-hMet30 mice, amyloid deposition started at 9 months of age, although the serum level of human mutant TTR reached the adult level at 1 month. These results suggest that intrinsic environmental factors other than the mutant gene are involved in the late-onset deposition of amyloid fibrils. Transgenic mice described here should be useful for analysing such factors

Role of serum amyloid P component for systemic amyloidosis in transgenic mice carrying human mutant transthyretin gene

The role of serum amyloid P component (SAP) for systemic amyloidosis in a transgenic mouse model for an autosomal dominant disease, familial amyloidotic polyneuropathy (FAP), was examined. For this purpose, two lines of transgenic mouse were produced by introducing the human mutant transthyretin (TTR) gene and the human SAP gene, respectively. Two lines of transgenic mice were mated to produce double transgenic mice carrying both human mutant TTR gene and human SAP gene. The serum concentration of human SAP in these transgenic mice was about 42 micrograms/ml and was about equal to that in human control serum. In case of single transgenic mice carrying human mutant TTR gene, amyloid deposition starts at around 6 months of age, and the amount of amyloid deposition increases gradually with age. Amyloid deposition is observed in many tissues including heart, kidney and thyroid gland, where amyloid deposition is commonly observed in FAP patients. In double transgenic mice, onset, progression and tissue distribution of amyloid deposition were the same as those in single transgenic mouse. These results clearly suggest that SAP is not important for the initiation and progression of amyloid deposition.

Secondary Hypoplastic Anemia in Patients with Familial Amyloidotic Polyneuropathy

The anemia of patients with familial amyloidotic polyneuropathy (FAP) was evaluated. Anemia was seen in 32 (91%) of the 35 FAP patients, more often with progression of the disease. The incidence of macrocytic hypochromic anemia was the most common type (40%). In 14 autopsied and 2 biopsied cases, no amyloid deposition was detected in the bone marrow. Thirteen (81%) of the 16 FAP patients showed hypoplastic bone marrow. Bone marrow aspiration of 2 patients revealed a decreased ratio of erythrocytic/myelocytic cells. The plasma levels of vitamin B12 and folate were within normal ranges. Neither oral nor intravenous administration of iron had any effect on the anemia of FAP patients. Intravenous erythropoietin elevated blood hemoglobin levels and blood pressure in 2 patients. Orthostatic hypotension, one of the most common symptoms of FAP, was unexpectedly improved. Secondary hypoplastic anemia is common in FAP, but treatment of anemia in this disease using erythropoietin is promising.

Complicating systemic amyloidosis in dystrophic epidermolysis bullosa, recessive type

&NA; An autopsy case of dystrophic epidermolysis bullosa, recessive type, complicated by systemic secondary amyloidosis is described. The patient had developed multiple bullous lesions and erosions from birth, followed by repeated infection. At autopsy, chronic persistent inflammation was observed in the skin and in various visceral organs, accompanied by systemic amyloidosis. By the peroxidase‐antiperoxidase (PAP) method, amyloid deposits stained positively for anti‐AA‐ protein antiserum. In the present case, we concluded that the systemic amyloidosis was of the AA type, and developed secondarily to the chronic persistent inflammation in the prolonged course of dystrophic epidermolysis bullosa, recessive type.

Pathology of familial amyloidotic polyneuropathy with TTR Met 30 in Kumamoto, Japan

Seventeen autopsy and 12 sural nerve biopsy cases of familial amyloidotic polyneuropathy (FAP) with transthyretin (TTR) Met 30 were examined clinicopathologically. In the autopsy cases, amyloid deposits were prominent in the peripheral nerve tissues, autonomic nervous system, choroid plexus, cardiovascular system and kidneys. Amyloid involvement in the posterior and anterior roots of the spinal cord, spinal ganglia, thyroid and gastrointestinal tract were also frequent. In the sural nerve biopsy, degenerative changes corresponding in degree to the duration of the clinical course were observed in the endoneurium, and amyloid deposition occurred around the blood vessels. Electron microscopy revealed degenerative changes in the axon, myelin sheath and Schwann cells. The morphometric study showed decreased numbers of small‐caliber myelinated fibers during the early stage. TTR was confirmed immunohistochemically as a major component of amyloid deposits. In transgenic mice carrying the human mutant TTR gene, amyloid deposition was observed in various organs except in the peripheral nerves and choroid plexus. Liver transplantation therapy to FAP patients has been carried out and future follow‐up studies should investigate the effects of therapy.

A Potential Animal Model for Familial Amyloidotic Polyneuropathy Through Introduction of Human Mutant Transthyretin Gene Into Mice

Familial amyloidotic polyneuropathy (FAP) has been identified in a number of kindreds of the world. FAP is an autosomal dominant disorder characterized by extracellular deposition of fibrillar amyloid protein and by prominent peripheral nerve involvement (1–4). This protein is mainly composed of TTR with a substitution of methionine for valine at position 30 in the FAP type I, as reported in Japan, Sweden and Portugal (5–7). This amino acid substitution is thought to lead to amyloid deposition. The human TTR gene has been cloned and well characterized at the molecular level (8, 9). All the FAP patients so far examined carry one mutant gene (10, 11). However in patients with FAP, the age at onset varies from 20 to 45 years. In addition the clinical syndrome is variable even among kindreds with the same genetic defect. These data suggest involvement of factor(s) other than the single nucleotide mutation in the TTR gene. To elucidate this factor(s) and to examine the pathological process of amyloid deposition, we have produced transgenic mice by microinjecting the cloned human mutant TTR gene into fertilized eggs of C57BL/6 mice.