Silencing of murine transthyretin and retinol binding protein genes has distinct and shared behavioral and neuropathologic effects.

The murine genes encoding transthyretin (TTR) and retinol binding protein (RBP) were independently silenced by targeted disruption more than 10 years ago. Studies of both strains showed surprisingly little impact on either thyroid function or retinoid metabolism. Silencing TTR led to a relatively mild behavioral phenotype. In order to gain insight into the behavioral effect and determine if it was related to TTR's function as the carrier of RBP we carried out simultaneous studies with homozygous Rbp4(-/-) and Ttr(-/-) animals 4-7 months of age. Both strains showed behavioral differences relative to Ttr and Rbp4 wild-type animals and each other. The patterns were discrete for each knockout although there was some overlap. Neuropathologic examination of the cortex and hippocampus revealed cortical and hippocampal (CA3) neuronal loss in both and some degree of gliosis, more pronounced in the Rbp4(-/-) mice. There also appeared to be a major reduction in proliferating neuroblasts in the subventricular zone in both strains, which was also more severe in the Rbp4(-/-) mice. This is the first description of behavioral abnormalities in Rbp4(-/-)mice. The data also indicate that it is unlikely that the behaviors seen in Ttr(-/-) mice are related to its function as an RBP carrier.

The V122I cardiomyopathy variant of transthyretin increases the velocity of rate-limiting tetramer dissociation, resulting in accelerated amyloidosis.

The transthyretin (TTR) amyloid diseases are of keen interest, because there are >80 mutations that cause, and a few mutations that suppress, disease. The V122I variant is the most common amyloidogenic mutation worldwide, producing familial amyloidotic cardiomyopathy primarily in individuals of African descent. The substitution shifts the tetramer-folded monomer equilibrium toward monomer (lowers tetramer stability) and lowers the kinetic barrier associated with rate-limiting tetramer dissociation (pH 7; relative to wild-type TTR) required for amyloid fibril formation. Fibril formation is also accelerated because the folded monomer resulting from the tetramer-folded monomer equilibrium rapidly undergoes partial denaturation and self-assembles into amyloid (in vitro) when subjected to a mild denaturation stress (e.g., pH 4.8). Incorporation of the V122I mutation into a folded monomeric variant of transthyretin reveals that this mutation does not destabilize the tertiary structure or alter the rate of amyloidogenesis relative to the wild-type monomer. The increase in the velocity of rate-limiting tetramer dissociation coupled with the lowered tetramer stability (increasing the mol fraction of folded monomer present at equilibrium) may explain why V122I confers an apparent absolute anatomic risk for cardiac amyloid deposition.

Abnormal immunoglobulin synthesis in monoclonal immunoglobulin light chain and light and heavy chain deposition disease.

The Congo red-binding fibrils of AL amyloidosis are the most common form of monoclonal immunoglobulin tissue deposition (MIDD). Nonetheless, the less structured deposits found in light chain deposition disease (LCDD) and the similar, but distinct, deposits of light and heavy chain deposition disease (LHCDD) and heavy chain deposition disease (HCDD) can produce significant clinical pathology. Analyses of immunoglobulin synthesis by bone marrow cells obtained from 7 patients with LCDD and LHCDD demonstrated the production of excess light chains in all and the presence of incomplete light chains or heavy chain fragments in 5, regardless of the presence of an intact monoclonal protein or related subunit in the serum or urine. Our data indicate that, as is the case with the fibrillar deposits of AL amyloid, the non-fibrillar forms of monoclonal Ig deposition (LCDD and LHCDD) can be associated with the presence of immunoglobulin fragments in bone marrow cells. In some instances these appeared to be synthetic in origin, although rapid intracellular proteolysis or a combination of both could not be excluded. In either case the fragments may be more susceptible to tissue deposition, with subsequent organ compromise, than intact Ig chains.

Infiltrative nonamyloidotic monoclonal immunoglobulin light chain cardiomyopathy: an underappreciated manifestation of plasma cell dyscrasias.

BACKGROUND: Infiltrative cardiomyopathies are characterized by diastolic dysfunction. In monoclonal plasma cell dyscrasias, organ compromise may be produced by tissue deposition of monoclonal immunoglobulins or their constituent peptides independently of the effects of unbridled plasma cell proliferation. The deposits may be fibrillar, as in light chain amyloid (AL) or nonfibrillar, as in light chain deposition disease (LCDD). AL disease of the heart is a restrictive cardiomyopathy. We hypothesized that, despite differences in physical properties, nonamyloidotic light chain deposition in the myocardium could produce similar clinical and physiological abnormalities. METHODS: Cardiac tissue from five patients with LCDD and cardiac dysfunction was examined by immunohistochemical and electron microscopic techniques. Hospital charts, electrocardiograms, echocardiograms and cardiac catheterization results were reviewed. In two cases, the original echocardiograms were reanalyzed. RESULTS: The five patients with nonamyloidotic light chain deposits in the myocardium had either mechanical or electrocardiographic abnormalities. In four with adequate clinical documentation, the diastolic dysfunction and conduction abnormalities were similar or identical to that described in cardiac AL disease. CONCLUSIONS: Although nonamyloidotic immunoglobulin light chain deposits in the myocardium differ in distribution and ultrastructural organization from the fibrillar deposits of AL disease, an analogous pattern of diastolic dysfunction and conduction disturbances results. The diagnosis should be considered in patients with a plasmacytic dyscrasia and restrictive cardiomyopathy in whom Congo red staining of endomyocardial biopsy tissue is negative. The diagnosis can be established by using the appropriate immunohistochemical and ultrastructural tissue examinations.

The genetics of the amyloidoses.

The amyloidoses are diseases in which abnormalities in the secondary structure of precursor proteins result in decreased solubility under physiologic conditions, with subsequent organ compromise. A total of 18 proteins have been definitively identified as amyloid precursors associated with human disease. Mutations in the genes that encode some of these proteins produce autosomal dominant disease in mid to late adult life. Until recently, the late onset has obscured the familial nature of some of the disorders. This is especially true in the apparently sporadic disease-producing deposits found even later in life. In many instances, these deposits are derived from precursors encoded by wild-type genes (perhaps influenced by alleles that are polymorphic in the normal population); in other cases, they represent autosomal dominant disease with age-dependent penetrance. The genetic aspects of amyloid diseases produced by the deposition of four different proteins have been investigated in detail and provide insights into the particular diseases and amyloidogenesis in general.

Familial amyloidosis in one Chinese family: clinical, immunological, and molecular genetic analysis.

Three members of a Taiwanese kindred developed severe, systemic, early onset (< age 25 years), biopsy-proven amyloidosis. Clinical features included upper and lower extremity sensorimotor neuropathy, abdominal pain, vomiting, corneal ulcerations, cardiomyopathy, and syncope. Immunohistochemical analysis indicated that the deposits consisted of transthyretin. Molecular genetic studies revealed a heterozygous codon 55 point mutation, resulting in a proline for leucine-substitution, a mutation previously associated with aggressive familial amyloidosis in a US kindred of Dutch and German descent. The clinical courses and echocardiographic findings are typical for many types of amyloidosis; the pathologic data and genetic studies were necessary to establish a precise diagnosis.

Variant-sequence transthyretin (isoleucine 122) in late-onset cardiac amyloidosis in black Americans.

BACKGROUND: After the age of 60, isolated cardiac amyloidosis is four times more common among blacks than whites in the United States; 3.9 percent of blacks are heterozygous for an amyloidogenic allele of the normal serum carrier protein transthyretin in which isoleucine is substituted for valine at position 122 (Ile 122). We hypothesized that the high prevalence of transthyretin Ile 122 is at least partially responsible for the increased frequency of senile cardiac amyloidosis among blacks. METHODS: Paraffin blocks of cardiac tissue were obtained from an earlier study of 52,370 autopsies in Los Angeles and were examined by immunohistochemical and DNA analyses. Samples were available from 32 of 55 blacks and 20 of 78 whites over 60 years of age with isolated cardiac amyloidosis and from two control groups (228 cases). RESULTS: Transthyretin amyloidosis was identified in 31 of the 32 cardiac-tissue samples from the black patients and in 19 of the 20 samples from the white patients. Six of the 26 analyzable DNA samples (23 percent) from the black patients and none of the 19 samples from the white patients were heterozygous for the Ile 122 variant. Four of 125 DNA samples obtained at autopsy (3.2 percent) from a second, more recent, age-matched cohort of blacks without amyloidosis at the same institution were heterozygous for the transthyretin Ile 122 allele. On reexamination the cardiac tissue from these four patients contained small amounts of amyloid not detected at the initial autopsies. All subjects with the Ile 122 variant had ventricular amyloid. CONCLUSIONS: The assessment of elderly black patients with unexplained heart disease should include a consideration of transthyretin amyloidosis, particularly that related to the Ile 122 allele.

Transthyretin Ile 122 and cardiac amyloidosis in African-Americans. 2 case reports.

Two cases of cardiac amyloidosis resulting from deposition of the Ile 122 variant of transthyretin in African-Americans are presented. These cases illustrate several typical features of this disorder, including electrocardiographic abnormalities and digoxin toxicity. Transthyretin Ile 122 is a common amyloidogenic variant in African-Americans (present as a heterozygous variant in 4% of this population); therefore, the diagnosis of transthyretin Ile 122 cardiac amyloidosis should be considered in African-Americans with unexplained restrictive cardiomyopathy or arrhythmias.

Revised transthyretin Ile 122 allele frequency in African-Americans.

The transthyretin (TTR) Ile 122 variant is associated with cardiac amyloidosis in individuals of African descent. To determine the prevalence of the allele encoding TTR Ile 122 in African-Americans, we have used PCR and restriction analysis to test DNA from African-Americans from various geographic areas, and found an allele frequency of 66/3376 (0.020), which is higher than the value we previously reported in a much smaller pilot study. Our data indicate that this TTR variant is present at equal frequency in African-Americans throughout the U.S., and suggest that this mutation may be a common, often unrecognized cause of cardiac disease in African-Americans.

Transthyretin Ser 6 gene frequency in individuals without amyloidosis.

Transthyretin (TTR) Ser 6 was originally described in a Scottish kindred without amyloidosis. This variant, arising from a G-->A transition in codon 6 that destroys an MspI site and creates a BsrI site, was present in none of 50 controls, and was therefore thought to be rare. This variant has subsequently been found in a normal human cDNA liver library and in two unrelated patients with familial amyloidosis and other TTR variants, raising the question whether it is actually a common polymorphism. To address this question, we performed PCR and restriction digestion of 574 DNA samples from people without evidence of amyloidosis or a known family history of amyloidosis. The TTR Ser 6 allele frequency was 33/558 (.060) in Caucasians (including 8/192 (.04) in North American Ashkenazic Jews, 16/218 (.07) in North American non-Jews, and 9/148 (.06) in Portuguese), 3(242 (.01) in African Americans, 0/140 in Africans, and 0/208 in Asians. These data are most suggestive of a single Caucasian founder and the known 25% admixture of "Caucasian" genes in the African-American population. Alternatively, as this variant arose from a transition at a CG dinucleotide "hot spot," it may have arisen on multiple occasions. These data indicate that TTR Ser 6 is a common non-amyloidogenic population polymorphism in Caucasians.

A double-variant transthyretin allele (Ser 6, Ile 33) in the Israeli patient "SKO" with familial amyloidotic polyneuropathy.

Transthyretin (TTR) isolated from amyloid fibrils from an Israeli patient ("SKO") with familial amyloidotic polyneuropathy has been studied by two groups of investigators. Originally, a position 49 Thr-->Gly substitution was reported; subsequently, a position 33 Phe-->Ile substitution was found instead. We have studied DNA from this patient by single strand conformation polymorphism analysis, restriction analysis, and DNA sequencing. On one allele, exon 2 contained both a T-->A transversion at the first position of codon 33, encoding the previously described Phe-->Ile substitution, and a G-->A transition at the first position of codon 6, encoding a Gly-->Ser substitution. The originally reported position 49 mutation was not encoded in the genomic DNA. This is the first report of a TTR double-variant allele in a patient with TTR amyloidosis.

Transthyretin Pro 36 associated with familial amyloidotic polyneuropathy in an Ashkenazic Jewish kindred.

Mutations in the serum protein transthyretin (TTR) cause amyloidosis involving the peripheral nerves, heart, and other organs. In Ashkenazic Jews, the only TTR variant described to date has been TTR Ile 33. We have studied DNA from another Ashkenazic Jewish kindred with familial amyloidotic polyneuropathy. Single-strand conformation polymorphism analysis, DNA sequencing, and restriction analysis indicated that this kindred has the TTR Pro 36 variant, previously described only in a Greek kindred.

Transthyretin Pro55, a variant associated with early-onset, aggressive, diffuse amyloidosis with cardiac and neurologic involvement.

Mutations in the protein transthyretin cause amyloidosis involving the heart, peripheral nerves, and other organs. A family from West Virginia developed an unusually aggressive form of widespread transthyretin amyloidosis. Single-strand conformation polymorphism analysis revealed a variant in the transthyretin gene, which was found on sequencing to be a T----C transversion at position 2 of codon 55, corresponding to a Leu----Pro substitution. The variant sequence was confirmed by restriction analysis and polymerase chain reaction (PCR)-primer introduced restriction analysis.

Oncogene alterations in rat colon tumors induced by N-methyl-N-nitrosourea.

The authors assayed oncogene alterations in rat colon tumors induced by the direct-acting chemical carcinogen, N-methyl-N-nitrosourea (MNU). DNA isolated from 34 adenomas and eight carcinomas, as well as adjacent normal colon, of 11 rats was assayed by Southern blotting for restriction fragment length polymorphisms and gene amplifications and deletions in 13 oncogenes known to be involved in human or other animal tumors. In addition to finding apparent point mutations or other small alterations in the fos and abl genes in individual rat colon tumors, the authors observed what appear to be larger alterations (ie, rearrangements, or intragenic insertions or deletions) in the H-ras and myb loci in several tumors. In contrast, no changes in the K-ras, N-ras, myc, N-myc, neu, raf, fms, met, and hst genes were seen in any of these tumors. The frequency of myb gene alterations was higher in carcinomas than in adenomas, suggesting that these changes occurred relatively late during tumorigenesis and were not direct effects of the carcinogen. In addition, the finding of alterations in two or three oncogenes in several MNU-induced rat colon tumors suggests the possibility of more widespread genomic lesions in this model.

Detection of transforming oncogenes in rat colon tumors induced by direct perfusion with N-methyl-N-nitrosourea.

We assayed rat colon tumors induced by N-methyl-N-nitrosourea (MNU) for transforming oncogenes by the NIH 3T3 transfection and nude mouse tumorigenicity assays. Transfection of DNA from 3 of 3 adenomas and 3 of 5 carcinomas induced transformed foci on NIH 3T3 cells. DNA from 2 of 3 primary foci also possessed focus-forming activity, and rat-specific sequences were observed in secondary focus DNAs. Furthermore, NIH 3T3 cells transfected with DNA from a carcinoma and from a primary focus derived from it, both positive in the focus-forming assay, induced tumors in nude mice. We found no evidence for rat H-ras, K-ras, or N-ras sequences in the DNA of any of 16 primary foci derived from 6 rat tumors; thus, in contrast to other animal tumor models induced by MNU, activation of the ras genes does not appear to predominantly occur in MNU-induced rat colon tumors. We also did not observe, in any of these foci, sequences corresponding to the rat neu, raf, fms, met, or hst genes, thus indicating that none of these is the transforming oncogene in our model. These results suggest that an as yet unidentified transforming oncogene may be activated in rat colon tumors induced by MNU.

Frequency and genetic background of the position 122 (Val----Ile) variant transthyretin gene in the black population.

Transthyretin (TTR) (122 Val----Ile), caused by a point mutation which destroys a MaeIII restriction site, is associated with cardiac amyloidosis in black individuals. To estimate the frequency of the MaeIII(-) gene in the black population without overt cardiac disease, DNA from 177 black individuals without amyloidosis was amplified by the PCR around TTR codon 122 and was digested with MaeIII. The MaeIII(-) gene frequency was 4/354 (1.1%; 95% confidence interval 0.32%2.7%), suggesting that the variant is relatively common in blacks. HLA genotype testing did not suggest that the TTR (122 Val----Ile) heterozygotes were of a closely related genetic background.