A novel apolipoprotein A-1 variant, Arg173Pro, associated with cardiac and cutaneous amyloidosis

An American kindred was found to have hereditary amyloidosis with cutaneous and cardiac involvement. Characterization of fibrils isolated from skin identified the amyloid protein as the N-terminal 90 to 100 residues of apolipoprotein A-1. Sequence of the apolipoprotein A-1 gene was normal except for a G/C transversion at position 1638 which predicts an Arg to Pro substitution at residue 173. This mutation, unlike previously described amyloidogenic mutations is not in the N-terminal fragment which is incorporated into the fibril. The mutation is at the same residue as in apolipoprotein A-1 Milano (Arg173Cys) which does not result in amyloid formation. Decreased plasma HDL cholesterol levels in carriers of the Arg173Pro mutation suggest an increased rate of catabolism as has been shown for the amyloidogenic Gly26Arg mutation. This suggests that altered metabolism caused by the mutation may be a significant factor in apolipoprotein A-1 fibrillogenesis.     

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

Summary 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. Singlestrand 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.     

A homozygous transthyretin variant associated with senile systemic amyloidosis: evidence for a late-onset disease of genetic etiology.

Senile systemic amyloidosis (SSA) is a late-onset disease characterized by deposition of amyloid fibrils containing transthyretin (TTR). Amino acid sequencing of protein isolated from the amyloid fibrils of a patient with SSA identified TTR containing a position - 122 isoleucine-for-valine substitution. This change led to the prediction of a genomic G-to-A transition, destroying an MaeIII restriction site. We confirmed the presence of the variant DNA fragment both by Southern blotting and by visualization of MaeIII digests of DNA amplified around codon 122, by using the polymerase chain reaction. The patient's DNA was entirely resistant to MaeIII cleavage; therefore, only the mutant sequence was present. DNA from none of either 24 controls or six other SSA patients contained the variant. Quantitative Southern blotting demonstrated that the patient's DNA contained two copies of the TTR gene per genome; the mutation was therefore homozygous rather than hemizygous. In the present case, the homozygous mutation TTR (122 Val----Ile) is associated with SSA, a finding which is consistent with autosomal recessive inheritance of this condition.     

Circulating proalbumin associated with a variant proteinase inhibitor

The unique finding of normal proalbumin in human plasma provides an insight into the mechanism of propeptide cleavage. Proalbumin, present as 1–5% of the total albumin, was found in a boy whose prime problem was the presence of a mutant proteinase inhibitor, α₁-antitrypsin Pittsburgh (358Met→Arg) [2]. The infeerred structure of human proalbumin was confirmed as ArgGlyValPheArgArgAlb. On incubation with various enzymes (trypsin, tryptase, thrombin, chymotrypsin, chymase and cathepsin B), only trypsin was capable of converting proalbumin to albumin. There was no conversion when proalbumin was incubated with whole blood, plasma or serum. However, intravenous injection of proalbumin into a rat resulted in complete conversion to albumin, the half-life of this process being 6 h. We conclude that propeptide cleavage is dependent on a serine proteinase which is inhibited intracellularly, by the mutant inhibitor, and that all the albumin in the boy was secreted as proalbumin, but was subjected to a separate cleavage process after export from the hepatocyte.     

In vivo metabolism of a mutant apolipoprotein, apoA-IIowa, associated with hypoalphalipoproteinemia and hereditary systemic amyloidosis.

Apolipoprotein (apo) A-I is the major protein constituent of plasma high density lipoproteins (HDL). A kindred has been identified in which a glycine to arginine mutation at residue 26 in apoA-I is associated with hypoalphalipoproteinemia and hereditary systemic amyloidosis. We isolated the mutant protein, termed apoA-IIowa, from the plasma of an affected subject and studied its in vivo metabolism compared to that of normal apoA-I in two heterozygous apoA-IIowa subjects and two normal controls. Normal and mutant apoA-I were radioiodinated with 131I and 125I, respectively, reassociated with autologous plasma lipoproteins, and simultaneously injected into all subjects. Kinetic analysis of the plasma radioactivity curves demonstrated that the mutant apoA-IIowa was rapidly cleared from plasma (mean fractional catabolic rate [FCR] 0.559 day-1) compared with normal apoA-I (mean FCR 0.244 day-1) in all four subjects. The FCR of normal apoA-I was also substantially faster in the heterozygous apoA-IIowa subjects (mean FCR 0.281 days-1) than in the normal controls (mean FCR 0.203 days-1). Despite the rapid removal from plasma of apoA-IIowa, the cumulative urinary excretion of its associated radioactivity after 2 weeks (44%) of the injected dose) was substantially less than that associated with normal apoA-I (78% of injected dose), indicating extravascular sequestration of radiolabeled apoA-IIowa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Visceral leishmaniasis with cardiac involvement in a dog: a case report

A dog presented with cutaneous nodules, enlarged lymph nodes and oedema in limbs, face and abdomen. The diagnosis of visceral leishmaniasis was established by identification of Leishmania amastigotes within macrophages from skin and popliteal lymph node biopsies. At necropsy, lesions were found in different organs, but it was particularly striking to observe large areas of pallor in the myocardium. Histological examination revealed an intense chronic inflammatory reaction in many organs, and numerous macrophages were found to contain amastigote forms of Leishmania. The inflammatory reaction was especially severe in the heart, where large areas of the myocardium appeared infiltrated with huge numbers of mononuclear immune cells, causing cardiac muscle atrophy and degeneration. Despite the severe inflammation, the number of parasitized macrophages was low in the myocardium, as revealed by immunohistochemical staining of Leishmania amastigotes. Because cardiac involvement is not usually described in this condition, this dog represents a very rare case of canine visceral leishmaniasis with affection of the myocardium.     

Conformational analysis of the riboflavin-responsive ETF:QO-p.Pro456Leu variant associated with mild multiple acyl-CoA dehydrogenase deficiency

Multiple-CoA dehydrogenase deficiency (MADD) is an inborn disorder of fatty acid and amino acid metabolism caused by mutations in the genes encoding for human electron transfer flavoprotein (ETF) and its partner electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). Albeit a rare disease, extensive newborn screening programs contributed to a wider coverage of MADD genotypes. However, the impact of non-lethal mutations on ETF:QO function remains scarcely understood from a structural perspective. To this end, we here revisit the relatively common MADD mutation ETF:QO-p.Pro456Leu, in order to clarify how it affects enzyme structure and folding. Given the limitation in recombinant expression of human ETF:QO, we resort to its bacterial homologue from Rhodobacter sphaeroides (Rs), in which the corresponding mutation (p.Pro389Leu) was inserted. The in vitro biochemical and biophysical investigations of the Rs ETF:QO-p.Pro389Leu variant showed that, while the mutation does not significantly affect the protein α/β fold, it introduces some plasticity on the tertiary structure and within flavin interactions. Indeed, in the p.Pro389Leu variant, FAD exhibits a higher thermolability during thermal denaturation and a faster rate of release in temperature-induced dissociation experiments, in comparison to the wild type. Therefore, although this clinical mutation occurs in the ubiquinone domain, its effect likely propagates to the nearby FAD binding domain, probably influencing electron transfer and redox potentials. Overall, our results provide a molecular rational for the decreased enzyme activity observed in patients and suggest that compromised FAD interactions in ETF:QO might account for the known riboflavin responsiveness of this mutation.     

Bone scintigraphy for cardiac amyloidosis imaging: Past,present and future

The aim of the article was to review the history of the emergence of ^99mTc-labeled phosphate agents in the exploration of cardiac amyloidosis and the give an overview of the technique, its accuracy and its application in clinical practice. Potential future directions are mentioned.     

Purification and characterization of GP-55, a protein associated with actin-based cytoplasmic gels derived from brain tissue

Incubation of rat brain cytoplasmic extracts results in the formation of three-dimensional gels that can be collapsed by low-speed centrifugation. Electrophoresis in polyacrylamide gels indicate that these cytoplasmic gels are composed of actin and several associated proteins. Among the latter we have identified a component with an apparent molecular mass of 55,000 daltons. The results of two-dimensional electrophoresis, peptide mapping, and in vivo labeling experiments with [35S]methionine, indicate that the 55-kDa protein is composed of two different gene products we have named alpha and beta; a third polypeptide, named beta', probably derives from the beta polypeptide as a result of some posttranslational modification(s). These three polypeptides, which have the same molecular weight but different isoelectric points, are present in a rather pure preparation of the 55-kDa protein, obtained from cytoplasmic gels by ion-exchange chromatography. In vitro, purified 55-kDa protein interacts in a specific manner with F-actin, as shown by viscosimetry and electron microscopy, leading to the formation of a complex network of cross-linked microfilament bundles. The results of immunofluorescence experiments indicate that the 55-kDa protein is located intracellularly in structures belonging to the Golgi apparatus, and that it is not only present in every type of rat cell tested but also in primary cultures of chick embryo neurons. Based on its intracellular location, we have named the 55-kDa protein as Golgi Protein-55 (GP-55).     

Antithrombin Cambridge, 384 Ala to Pro: a new variant identified using the polymerase chain reaction

An antithrombin III variant was identified in the plasma of a female patient with a history of recurrent thromboses. The variant was shown to have normal antigenic levels but reduced heparin and progressive inhibitory activity consistent with an abnormality affecting function at the reactive centre. Polymerase chain reaction amplification of exon 6 of the gene with direct sequencing showed a point mutation resulting in the substitution of a proline for alanine at position 384. This substitution will predictably alter the conformation of the peptide loop containing the reactive centre of the molecule.     

JC virus encephalopathy is associated with a novel agnoprotein-deletion JCV variant

JC virus encephalopathy (JCVE) is a newly described gray matter disease of the brain caused by productive infection of cortical pyramidal neurons. We characterized the full length sequence of JCV isolated from the brain of a JCVE patient, analyzed its distribution in various compartments by PCR, and determined viral gene expression in the brain by immunohistochemistry(IHC). We identified a novel JCV variant, JCV(CPN1), with a unique 143 bp deletion in the Agno gene encoding a truncated 10 amino acid peptide, and harboring an archetype-like regulatory region. This variant lacked one of three nuclear protein binding regions in the Agno gene. It was predominant in the brain, where it coexisted with an Agno-intact wild-type strain. Double immunostaining with anti-Agno and anti- VP1 antibodies demonstrated that the truncated JCV(CPN1) Agno peptide was present in the majority of cortical cells productively infected with JCV. We then screened 68 DNA samples from 8 brain, 30 CSF and 30 PBMC samples of PML patients, HIV+ and HIV- control subjects. Another JCV(CPN) strain with a different pattern of Agno-deletion was found in the CSF of an HIV+/PML patient, where it also coexisted with wild-type, Agno-intact JCV. These findings suggest that the novel tropism for cortical pyramidal neurons of JCV(CPN1), may be associated with the Agno deletion. Productive and lytic infection of these cells, resulting in fulminant JCV encephalopathy and death may have been facilitated by the co-infection with a wild-type strain of JCV.     

Differences in planning performance,a neurocognitive endophenotype,are associated with a functional variant in PER3 gene

Performance alterations in executive function have been studied as potential endophenotypes for several neuropsychiatric diseases. Planning is an important component of executive function and has been shown to be affected in diseases such as attention deficit hyperactivity disorder, schizophrenia, obsessive–compulsive disorder and Parkinson’s disease. Several genes related to dopaminergic systems, such as COMT, have been explored as candidates for influencing planning performance. The circadian clock gene PERIOD3 (PER3) has been shown to be associated with several complex behaviors in humans and could be involved in different signaling mechanisms. In this study, we evaluated the possible association between a functional polymorphism in the PER3 gene (PER3-VNTR, rs57875989) and performance in a commonly used test of planning (Tower of London, TOL) in 229 healthy subjects from Bogotá, Colombia. PER3-VNTR genotyping was carried out with conventional PCR and all participants completed the TOL test using the computerized Psychology Experiment Building Language (PEBL) battery. A linear regression model was used for the analysis of association with the SNPStats program. We found that 4/4 genotype carriers showed a better performance and made fewer moves, in comparison to 4/5 and 5/5 genotype carriers (p?=?0.003). These results appear to be independent from effects of this polymorphism on self-reported average hours of sleep during work days in our sample. This is the first evidence of an association between PER3-VNTR and planning performance in a sample of healthy subjects and our results are consistent from previous findings for alterations in other cognitive domains. Future studies examining additional genes could lead to the identification of novel molecular underpinnings of planning in healthy subjects and in patients with neuropsychiatric disorders.     

Meningoencephalitis associated with passive immunization of a transgenic murine model of Alzheimer's amyloidosis

Immunization against the Abeta peptide reverses the pathologic and behavioral manifestations of Alzheimer's disease in murine models. Since active immunization is associated with an autoimmune meningoencephalitis in a subset of humans, passive transfer of anti-Abeta immunoglobulin is being pursued as a potentially safer alternative. We have identified cases of meningoencephalitis subsequent to peripheral and intracerebral passive immunization of Tg2576 mice. The vasocentric mononuclear infiltrate localized only to brain regions affected by Abeta amyloid deposits suggesting that the inflammatory reaction was Abeta specific. This report indicates that current passive immunization in humans should proceed with careful regard for autoimmune complications.     

Identification of S-sulfonation and S-thiolation of a novel transthyretin Phe33Cys variant from a patient diagnosed with familial transthyretin amyloidosis

Familial transthyretin amyloidosis (ATTR) is an autosomal dominant disorder associated with a variant form of the plasma carrier protein transthyretin (TTR). Amyloid fibrils consisting of variant TTR, wild-type TTR, and TTR fragments deposit in tissues and organs. The diagnosis of ATTR relies on the identification of pathologic TTR variants in plasma of symptomatic individuals who have biopsy proven amyloid disease. Previously, we have developed a mass spectrometry-based approach, in combination with direct DNA sequence analysis, to fully identify TTR variants. Our methodology uses immunoprecipitation to isolate TTR from serum, and electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry (MS) peptide mapping to identify TTR variants and posttranslational modifications. Unambiguous identification of the amino acid substitution is performed using tandem MS (MS/MS) analysis and confirmed by direct DNA sequence analysis. The MS and MS/MS analyses also yield information about posttranslational modifications. Using this approach, we have recently identified a novel pathologic TTR variant. This variant has an amino acid substitution (Phe --> Cys) at position 33. In addition, like the Cys10 present in the wild type and in this variant, the Cys33 residue was both S-sulfonated and S-thiolated (conjugated to cysteine, cysteinylglycine, and glutathione). These adducts may play a role in the TTR fibrillogenesis.