Early data on long-term efficacy and safety of inotersen in patients with hereditary transthyretin amyloidosis: a 2-year update from the open-label extension of the NEURO-TTR trial.

BACKGROUND AND PURPOSE: Hereditary transthyretin (hATTR) amyloidosis causes progressive polyneuropathy resulting from transthyretin (TTR) amyloid deposition throughout the body, including the peripheral nerves. The efficacy and safety of inotersen, an antisense oligonucleotide inhibitor of TTR protein production, were demonstrated in the pivotal NEURO-TTR study in patients with hATTR polyneuropathy. Here, the long-term efficacy and safety of inotersen are assessed in an ongoing open-label extension (OLE) study. METHODS: Patients who completed NEURO-TTR were eligible to enroll in the OLE (NCT02175004). Efficacy assessments included the modified Neuropathy Impairment Score plus seven neurophysiological tests composite score (mNIS + 7), the Norfolk Quality of Life - Diabetic Neuropathy (Norfolk QOL-DN) questionnaire total score and the Short-Form 36 Health Survey (SF-36) Physical Component Summary (PCS) score. Safety and tolerability were also assessed. RESULTS: Overall, 97% (135/139) of patients who completed NEURO-TTR enrolled in the OLE. Patients who received inotersen for 39 cumulative months in NEURO-TTR and the OLE continued to show benefit; patients who switched from placebo to inotersen in the OLE demonstrated improvement or stabilization of neurological disease progression by mNIS + 7, Norfolk QOL-DN and SF-36 PCS. No new safety concerns were identified. There was no evidence of increased risk for grade 4 thrombocytopenia or severe renal events with increased duration of inotersen exposure. CONCLUSION: Inotersen slowed disease progression and reduced deterioration of quality of life in patients with hATTR polyneuropathy. Early treatment with inotersen resulted in greater long-term disease stabilization than delayed initiation. Routine platelet and renal safety monitoring were effective; no new safety signals were observed.

Hereditary renal amyloidosis associated with a mutant fibrinogen alpha-chain.

Three members of a family who died with renal amyloidosis were found to share a single nucleotide substitution in the fibrinogen alpha-chain gene. The predicted arginine to leucine mutation (Arg554Leu) was proven by amino acid sequence analysis of amyloid fibril protein isolated from postmortem kidney of an affected individual. Direct genomic DNA sequencing and restriction fragment length polymorphism analysis demonstrated that all three affected individuals had the guanine to thymine 4993 transversion. This is the first demonstration of hereditary amyloidosis associated with a variant fibrinogen alpha-chain. Variants of circulating fibrinogen may be the cause of a number of systemic amyloidoses with primarily renal involvement.

Pulmonary embolism in pregnancy. Consensus and controversies.

Venous thrombotic events (VTE) occur 1-2 per 10,000 pregnancies and remain one of the leading causes of maternal mortality in the developed world. The two largest risk factors are a personal history of VTE and heritable thrombophilias. D-dimer tests for VTE in pregnancy have a high false positive rate and at least some false negatives have been reported. Compression ultrasound should be used to evaluate pregnant women for deep venous thrombosis followed by magnetic resonance imaging of the pelvis for a negative test and strong remaining clinical suspicion. For pulmonary embolism, a chest x-ray should be used to triage the patient to either a ventilation/perfusion study after a normal X-ray or a CT pulmonary angiogram after an abnormal one. Treatment generally consists of low molecular weight heparin through a minimum of six weeks post-partum. Thombolysis might have merit in life-threatening, massive pulmonary embolism. VTE prophylaxis in at-risk populations remains a major area of uncertainty. Mechanical prophylaxis for all women undergoing cesarean, in particular, has a paucity of supportive evidence.

Isolation and identification by sequence analysis of experimentally induced guinea pig amyloid fibrils.

Amyloidosis was produced experimentally in guinea pigs by multiple casein injections. Amyloid fibrils were isolated and fractionated and a protein obtained that had an amino acid composition comparable with A protein, a unique nonimmunoglobulin constituent of secondary amyloid deposits. N-terminal sequence analysis demonstrated a sequence homologous with that of A proteins from human and monkey preparations but preceded by a 5-residue peptide which had an N-terminal histidine. A definite species specificity in A protein from human and guinea pig was identified on immunologic analysis.

Suppression of in vitro antibody response by a serum factor (SAA) in experimentally induced amyloidosis.

Serum from CBA/J mice made amyloidotic by chronic casein injections has been shown to suppress in vitro antibody response to SRBC. Similar suppression was also found with normal mouse serum but to a much lesser degree. This suppressive activity of both amyloidotic serum and normal serum was removed by absorption of the sera with antiserum to protein AA, the major constituent of casein-induced (secondary) amyloid fibrils. This antiserum to the amyloid fibril protein AA (mol wt 8,400 daltons) detects an immunologically cross-reacting serum alpha globulin (SAA) (mol wt approx. 100,000). It is postulated that the serum factor (SAA) is a regulator of antibody response and may be present in elevated amounts as the result of chronic antigenic stimulation.

Diagnosis and Screening of Patients with Hereditary Transthyretin Amyloidosis (hATTR): Current Strategies and Guidelines.

The outlook for transthyretin amyloidosis (ATTR) is changing with the availability of new and emerging treatments. ATTR now appears to be more common than previously thought and is no longer viewed as an obscure diagnosis with a grim prognosis. Now more than ever, there is growing emphasis on the need for early diagnosis because the treatments appear to be most effective if started in earlier stages of the disease. Diagnosing ATTR is a challenge as it may initially present with nonspecific symptoms and it is often thought of as a diagnosis of exclusion. Increased awareness is imperative as new treatments offer hope and have the potential to change the disease trajectory. ATTR commonly presents with neurological and cardiac features. Transthyretin (TTR) is a protein produced in the liver which misfolds either due to genetic mutations or due to aging and results in deposition of amyloid fibrils in organs and tissues. Apart from the traditional imaging modalities, newer techniques including echocardiographic strain imaging, magnetic resonance imaging (MRI), and nuclear scintigraphy, as well as the increased availability of genetic testing are aiding in making a timely diagnosis. In this review, we present the current understanding of the ATTR disease process, diagnostic and surveillance approaches, newer treatment modalities, and the future directions.

A mutation in the amyloid precursor protein associated with hereditary Alzheimer's disease.

Alzheimer's disease is a form of localized amyloidosis characterized by cerebral cortical amyloid plaques, neurofibrillary tangles, and amyloid deposits within the walls of leptomeningeal vessels. Although most cases of Alzheimer's disease are sporadic, kindreds with autosomal-dominant inheritance of the syndrome suggest that a single mutation may be important in pathogenesis. Direct sequencing of DNA from a family with autopsy-proven Alzheimer's disease revealed a single amino acid substitution (Phe for Val) in the transmembrane domain of the amyloid precursor protein. This mutation correlates with the presence of Alzheimer's disease in all patients in this study, and may be the inherited factor causing both amyloid fibril formation and dementia.

Autocrine induction of collagenase by serum amyloid A-like and beta 2-microglobulin-like proteins.

Two autocrine proteins of 14 and 12 kilodaltons that induce the synthesis of rabbit fibroblast collagenase were identified. The proteins were purified from serum-free culture medium taken from rabbit synovial fibroblasts stimulated with phorbol myristate acetate. The amino-terminal sequences of the 14- and 12-kilodalton species were approximately 60 to 80 percent homologous with serum amyloid A and beta 2 microglobulin, respectively. The polyacrylamide gel-eluted proteins retained the ability to induce collagenase synthesis in rabbit and human fibroblasts. These autocrine proteins may provide a means to modulate collagenase synthesis in normal remodeling as well as in inflammation and disease states.

Partial amino acid sequence homology between an heredofamilial amyloid protein and human plasma prealbumin.

Amyloid fibril protein has been isolated from the tissues of a patient of Swedish ancestry with autosomal dominant heredofamilial amyloidosis. After solubilization in guanidine HCl, a significant amount of the protein was contained in a homogeneous low molecular weight fraction. Molecular weight of approximately 14,000, amino acid analysis, double immunodiffusion analysis and immunoelectrophoresis all supported this material being a prealbumin-related protein. Automated sequence analysis gave a mixture of amino acids at each step, suggesting an heterogeneous NH2-terminus. After cleavage of the protein with cyanogen bromide, a homogeneous peptide was obtained with the sequence Val-Val-Val-Leu-Asp-Ala-Val-Arg-Gly-Thr-Pro- corresponding in 9 of the 11 positions analyzed with the known sequence of human prealbumin, starting with position 14. Antiserum raised to the amyloid protein reacted with normal human prealbumin. After absorption with normal human serum, this antiserum continued to detect a determinant in the amyloid patient's serum, suggesting an abnormal serum prealbumin, which may be the precursor of the fibril protein in this type of heredo-familial amyloidosis. Indirect immunohistochemical studies on kidney tissue from the patient with amyloidosis showed marked staining with anti-prealbumin and anti-heredofamilial amyloid protein, but not with anti-AA or anti-kappa antisera. No genetic association between this family and amyloidosis and Portuguese families with familial amyloid polyneuropathy is known.

Characterization of a transthyretin (prealbumin) variant associated with familial amyloidotic polyneuropathy type II (Indiana/Swiss).

Amyloid fibrils were isolated from cardiac tissue of two brothers who died from familial amyloidotic polyneuropathy (FAP) type II. Sequence analysis on peptides derived from proteolytic cleavage with trypsin and fragmentation with cyanogen bromide reveal that the fibril subunit protein is derived from plasma transthyretin (prealbumin). About two-thirds of the fibril subunit protein was found to contain an amino acid substitution at position 84 where the normal isoleucine residue has been replaced by serine. Sequence analysis of the plasma transthyretin (prealbumin) from the two brothers as well as two clinically diagnosed FAP type II family members and two of four children of affected individuals showed the presence of serine at position 84. The presence of this substitution also correlates with low serum levels of retinol-binding protein and thus transthyretin (prealbumin) position 84 may be involved with the interaction of these two proteins.

Identification of carriers of a variant plasma prealbumin (transthyretin) associated with familial amyloidotic polyneuropathy type I.

A method is described for detecting carriers of a variant plasma prealbumin that is associated with familial amyloidotic polyneuropathy (FAP) type I. It is based on the finding of an extra methionine in the variant prealbumin, at position 30 from the amino terminals. Since normal prealbumin has only one methionine (position 13), treatment with cyanogen bromide (CNBr), which cleaves only at methionines, results in two peptides. CNBr treatment of the variant prealbumin gives three peptides. The extra can then be detected in two ways: by HPLC using a reverse phase C18 column, and by sequential Edman degradation. Each method can detect as little as 1% variant prealbumin in isolated plasma prealbumin, and therefore, can identify carriers of the gene for the variant protein. Since FAP type I usually is not manifest until after the childbearing years, this method to identify carriers of the gene offers a new approach for genetic counseling of families with this disease. To date, kindreds with hereditary amyloidosis that could benefit from these studies include those with FAP type I of Swedish, Japanese, and Portuguese origins.

Hereditary renal amyloidosis with a novel variant fibrinogen.

Two families with hereditary renal amyloidosis were found to have a novel mutation in the fibrinogen A alpha chain gene. This form of amyloidosis is an autosomal dominant condition characterized by proteinuria, hypertension, and subsequent azotemia. DNAs of patients with amyloidosis were screened for a polymorphism in fibrinogen A alpha chain gene by single-strand conformation polymorphism analysis, and affected individuals from two kindreds were found to have a mutation. Both of these kindreds are American of Irish descent presenting with non-neuropathic, nephropathic amyloidosis in the fifth to the seventh decade of life. DNA sequencing showed a point mutation in the fibrinogen A alpha chain gene that is responsible for substitution of valine for glutamic acid at position 526. By restriction fragment length polymorphism analysis, 7 affected individuals and 14 asymptomatic individuals in these two kindreds were positive for the fibrinogen A alpha chain Val 526 gene. Fibrinogen was isolated from plasma of a heterozygous gene carrier and shown to contain approximately 50% variant fibrinogen. Discovery of this new mutation confirms the association between fibrinogen A alpha chain variant and hereditary renal amyloidosis and establishes a new biochemical subtype of amyloidosis.

Human serum amyloid A. Three hepatic mRNAs and the corresponding proteins in one person.

Serum amyloid A protein (SAA) is a major acute-phase protein in humans and most other mammals. In addition, it is the serum precursor of the major protein constituent of reactive amyloid fibrils. Sequence analyses have identified a number of polymorphic forms of human SAA and amyloid A protein (AA), but the question of the number of genes encoding SAA in the human has not been addressed. In addition, there are insufficient data to predict whether one form of SAA predisposes to amyloid fibril formation. In the present study three separate SAA proteins have been isolated from the plasma of one individual and completely sequenced. While two of the SAA forms (SAA2 alpha and SAA2 beta) differ from each other only at position 71, they differ from the most abundant form (SAA1) at seven and eight other positions, respectively. Nucleotide sequencing of cDNAs from a liver library of this individual identified all three mRNs coding for these proteins and proved that: (a) the often-reported absence of arginine at the amino terminus of SAA proteins must result from proteolytic processing of the protein; (b) the polymorphism involving histidine and arginine at position 71 is present at the DNA level and therefore is not due to an event at the translational level; (c) there are at least two genes coding for human SAA. Comparison of these data to published sequences of SAA and AA proteins may help in identifying genetically determined forms of SAA which predispose to reactive amyloid fibril formation.

Biochemical and molecular genetic characterization of a new variant prealbumin associated with hereditary amyloidosis.

Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant late-onset disorder characterized by the extracellular deposition of amyloid fibrils. In all cases studied these fibrils have been found to be composed of plasma prealbumin (transthyretin) containing a single amino acid substitution. Biochemical studies were conducted on amyloid from one patient and plasma prealbumin from his affected brother, both part of a large kindred from the Appalachian region of the United States. Sequence analysis of the amyloid subunit protein showed it to be prealbumin with about two-thirds of the molecules containing a substitution of alanine for threonine at position 60. Studies of the plasma prealbumin showed that the same substitution was present in 40-45% of the protein. Based on this substitution and the prealbumin cDNA sequence, a Pvu II restriction fragment length DNA polymorphism (RFLP) was predicted and demonstrated in DNA of both patients as well as other family members. This RFLP confirms the predicted DNA mutation responsible for the protein variant, and represents an accurate method for detection of this gene.

Kinetics of serum amyloid protein A in casein-induced murine amyloidosis.

Serum amyloid protein A (SAA), the precursor of secondary amyloid protein, is elevated in chronic diseases which are associated with an increased incidence of amyloid. However, SAA is also elevated in acute bacterial and viral infections and somes forms of cancer. The murine model of casein-induced amyloidosis was studied to determine the relationship between SAA production and amyloid deposition. SAA levels measured by radioimmunoassay were found to be as high as 200 times the normal level in CBA/J mice receiving daily parenteral casein. After a single injection of casein the SAA level was elevated by 3h and peaked by 12-18 h. Similar levels were found in casein-treated A/J mice, a strain less susceptible to the induction of amyloid. Parenterally administered bovine serum albumin, which has low potential for amyloid induction, gave SAA levels in CBA/J and A/J mice comparable to casein treatment. These data show that, while SAA levels are elevated during chronic antigenic stimulation, there are other factors involved in amyloid formation. These factors may include alterations in the degradation of SAA by the reticuloendothelial system caused by substances such as casein. Nude (athymic) mice were shown to attain high levels of SAA after receiving casein parenterally. Therefore, thymus-derived lymphocytes are not necessary for the synthesis of SAA.

Identification of a new hereditary amyloidosis prealbumin variant, Tyr-77, and detection of the gene by DNA analysis.

In the last several years, five human plasma prealbumin (transthyretin) variants have been discovered in association with hereditary amyloidosis, a late-onset fatal disorder. We recently studied a patient of German descent with peripheral neuropathy and bowel dysfunction. Biopsied rectal tissue contained amyloid that stained with anti-human prealbumin. Amino acid sequence analysis of the patient's plasma prealbumin revealed both normal and variant prealbumin molecules, with the variant containing a tyrosine at position 77 instead of serine. We predicted a single nucleotide change in codon 77 of the variant prealbumin gene, which we then detected in the patient's DNA using the restriction enzyme SspI and a specifically tailored genomic prealbumin probe. DNA tests of other family members identified several gene carriers. This is the sixth prealbumin variant implicated in amyloidosis, and adds to the accumulating evidence that the prealbumin amyloidoses are more varied and prevalent than previously thought.

A point mutation in transthyretin increases affinity for thyroxine and produces euthyroid hyperthyroxinemia.

In a family expressing euthyroid hyperthyroxinemia, an increased association of plasma thyroxine (T4) with transthyretin (TTR) is transmitted by autosomal dominant inheritance and is secondary to a mutant TTR molecule with increased affinity for T4. Eight individuals spanning three generations exhibited the abnormality. Although five of eight individuals had elevated total T4 concentrations, all affected individuals were clinically euthyroid and all had normal free T4 levels. Purified TTR from the propositus had an affinity for 125I-T4 three times that of control TTR. Exons 2, 3, and 4 (representing greater than 97% of the coding sequence) of the TTR gene of DNA prepared from the propositus' peripheral blood leukocytes were amplified using the polymerase chain reaction (PCR) and were sequenced after subcloning. Exons 2 and 3 were indistinguishable from normal. In 50% of clones amplified from exon 4, a substitution of adenine (ACC) for guanine (GCC) in codon 109 resulted in the replacement of threonine-for-alanine, a mutation confirmed by amino acid sequencing of tryptic peptides derived from purified plasma TTR. The adenine-for-guanine substitution abolishes one of two Fnu 4H I restriction sites in exon 4. PCR amplification of exon 4 of TTR and restriction digestion with Fnu 4H I confirmed that five affected family members with increased binding of 125I-T4 to TTR are heterozygous for the threonine 109 substitution that increases the affinity of this abnormal TTR for T4.

Familial amyloidotic polyneuropathy.

Amyloidosis has received considerable attention recently because of its association with Alzheimer's disease. Actually, the amyloid in the cortical plaques, which is characteristic of Alzheimer's disease, is a localized form of amyloid deposition. Although intracranial vascular amyloid deposits which contain the A4 or beta-protein are usually associated with Alzheimer's disease, deposition of this amyloid fibril substance in other organs of the body has not been described. Much less attention has been paid to amyloid involvement of the PNS which is a fascinating subject in itself and is the subject of this review.

Maternal collapse: Challenging the four-minute rule.

INTRODUCTION: The current approach to, cardiopulmonary resuscitation of pregnant women in the third trimester has been to adhere to the "four-minute rule": If pulses have not returned within 4min of the start of resuscitation, perform a cesarean birth so that birth occurs in the next minute. This investigation sought to re-examine the evidence for the four-minute rule. METHODS: A literature review focused on perimortem cesarean birth was performed using the same key words that were used in formulating the "four-minute rule." Maternal and neonatal injury free survival rates as a function of arrest to birth intervals were determined, as well as actual incision to birth intervals. RESULTS: Both maternal and neonatal injury free survival rates diminished steadily as the time interval from maternal arrest to birth increased. There was no evidence for any specific survival threshold at 4min. Skin incision to birth intervals of 1min occurred in only 10% of women. CONCLUSION: Once a decision to deliver is made, care providers should proceed directly to Cesarean birth during maternal cardiac arrest in the third trimester rather than waiting for 4min for restoration of the maternal pulse. Birth within 1min from the start of the incision is uncommon in these circumstances.

Characterization of amyloid A protein in human secondary amyloidosis: the predominant deposition of serum amyloid A1.

Serum amyloid A protein (SAA) is the plasma precursor for amyloid A protein (AA), the subunit protein in amyloid deposits of secondary or reactive amyloidosis. Several forms of acute phase SAA have been identified in human plasma. To elucidate whether one of these forms of SAA predominates in the formation of AA amyloid deposits, the amino acid sequence of the subunit protein in six cases of reactive amyloidosis was investigated. Minimal heterogeneity was present at the N-terminus as all samples started with residue 1, 2, or 3 of SAA. The C-terminus, however, was more heterogeneous with the AA protein in each case terminating at multiple sites from residue 58 to 84 of SAA. Since less than 20% of the AA protein in each case contained sequence past residue 67 of SAA, the sequence and recovery of tryptic peptides containing residues 52, 57, and 60 where human SAA1 and 2 differ was used to determine the relative amounts of SAA1 and 2 present. One sample contained only SAA1 sequence, four contained approx. 11% or less of SAA2 sequence, and the sixth contained 24-33% of SAA2 sequence. Thus, while five of the six AA samples contained both SAA1 and 2, the predominant form in all cases was SAA1. In three of the six cases, the protein defensin was isolated along with the AA protein from the fibrils. This may suggest neutrophil involvement in SAA processing to AA fibrils.