A novel application for 222Rn emanation standards: radon-cryptophane host chemistry.

In collaboration with the University of Pennsylvania, a (222)Rn emanation source was used for the determination of the binding affinity of radon to a cryptophane molecular host. This source was similar to a (222)Rn emanation standard that was developed and disseminated by the National Institute of Standards and Technology (NIST). The novel experimental design involved performing the reactions at femtomole levels, developing exacting gravimetric sampling methods and making precise (222)Rn assays by liquid scintillation counting. A cryptophane-radon association constant was determined, K(A)=(49,000±12,000) L mol(-1) at 293 K, which was the first measurement of radon binding to a molecular host.

Amyloid and nonfibrillar deposits in mice transgenic for wild-type human transthyretin: a possible model for senile systemic amyloidosis.

The human serum protein transthyretin (TTR) is highly fibrillogenic in vitro and is the fibril precursor in both autosomal dominant (familial amyloidotic polyneuropathy [FAP] and familial amyloidotic cardiomyopathy [FAC]) and sporadic (senile systemic amyloidosis [SSA]) forms of human cardiac amyloidosis. We have produced mouse strains transgenic for either wild-type or mutant (TTRLeu55Pro) human TTR genes. Eighty-four percent of C57BI/6xDBA/2 mice older than 18 months, transgenic for the wild-type human TTR gene, develop TTR deposits that occur primarily in heart and kidney. In most of the animals, the deposits are nonfibrillar and non-Congophilic, but 20% of animals older than 18 months that bear the transgene have human TTR cardiac amyloid deposits identical to the lesions seen in SSA. Amino terminal amino acid sequence analysis and mass spectrometry of the major component extracted from amyloid and nonamyloid deposits revealed that both were intact human TTR monomers with no evidence of proteolysis or codeposition of murine TTR. This is the first instance in which the proteins from amyloid and nonfibrillar deposits in the same or syngeneic animals have been shown to be identical by sequence analysis. It is also the first time in any form of amyloidosis that nonfibrillar deposits have been shown to systematically occur temporally before the appearance of fibrils derived from the same precursor in the same tissues. These findings suggest, but do not prove, that the nonamyloid deposits represent a precursor of the fibril. The differences in the ultrastructure and binding properties of the deposits, despite the identical sizes and amino terminal amino acid sequences of the TTR and the dissociation of deposition and fibril formation, provide evidence that in vivo factors, perhaps associated with aging, impact on both systemic precursor deposition and amyloid fibril formation.

Marked systemic amyloid angiopathy in patients with val 107 transthyretin mutation.

We report three non-inbred patients with Val 107 transthyretin (TTR) amyloidosis. Clinical features were remarkable by the combination of peripheral polyneuropathy, carpal tunnel syndrome, cardiomyopathy, and epilepsy. Pathologic examination disclosed unusual striking systemic amyloid angiopathy in all studied tissues including nerve, muscle, gut, lung, salivary glands, and synovial membrane. It appears that the rare TTR Val 107 variant causes a peculiar familial amyloid syndrome characterized by both widespread systemic TTR amyloidosis and central nervous system deposition sufficient to cause seizures, pointing out the extent of TTR amyloidosis phenotypic heterogeneity.

Transthyretin amyloidosis (serine 44) with headache, hearing loss, and peripheral neuropathy.

A 32-year-old man of Irish descent presented with severe progressive headache and sensorineural hearing loss. MRI/magnetic resonance angiography head scans were normal. A length-dependent sensorimotor peripheral neuropathy with autonomic dysfunction predated these symptoms. Systemic organ involvement and transthyretin (TTR) amyloid immunostaining of bone marrow and fat aspirate were documented. Direct DNA sequencing revealed both the normal TTT (phenylalanine) and a new variant TCT (serine) at position 44 of the TTR gene. This case expands the genotypic and phenotypic variability within TTR amyloidosis.

Familial amyloid with a transthyretin leucine 33 mutation presenting with ascites.

A 65-year-old female presented with symptomatic ascites. Light and electron microscopy examination of omental and peritoneal tissue obtained at exploratory laparotomy revealed amyloidosis. Immunochemical studies of the amyloid tissue showed positive staining with antibodies to transthyretin. Polymerase chain reaction (PCR), single strand conformation polymorphism analysis, and direct DNA sequencing demonstrated a transthyretin phenylalanine to leucine substitution at codon 33. This is only the second reported case of a transthyretin leucine 33 mutation. Moreover, this patient is unique among cases of transthyretin-associated amyloidosis with the clinical presentation of ascites.

Novel transthyretin missense mutation (Thr34) in an Italian family with hereditary amyloidosis.

We report on the genetic and molecular characterisation of an Italian family with a late-onset, autosomal dominant transthyretin amyloidosis. The transthyretin gene was analysed by polymerase chain reaction (PCR), restriction generating PCR, and sequencing, allowing us to discover in one allele a novel point mutation. It consists of a G to C transversion at position 1692 of the genomic sequence, leading to a Thr for Arg substitution at the position 34 of the polypeptidic chain. This mutation is associated with a severe sensory-motor peripheral neuropathy and a restrictive cardiomyopathy.

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.

Amyloid fibril composition and transthyretin gene structure in senile systemic amyloidosis.

BACKGROUND: In many different forms of amyloidosis, transthyretin (TTR) comprises the major amyloid fibril protein. In the familial forms, various TTR mutations are linked to disease. This study was designed to characterize the components of the TTR-derived amyloid fibril protein in senile systemic amyloidosis and to determine whether any mutation in the TTR gene was present. EXPERIMENTAL DESIGN: Heart tissues from two patients with advanced senile systemic amyloidosis were studied. Amyloid fibrils were extracted and the amyloid fibril protein purified. The relationship between full-length and fragmented TTR and the amino acid sequence of the TTR were determined. The TTR gene was studied by single-strand conformation polymorphism analysis or direct sequencing. RESULTS: In both cases, the amyloid deposits contained full-length TTR and a complex mixture of TTR fragments. The fragments, most of which had their N-termini at positions 46-52, predominated. No amino acid substitution was identified. The N-terminal fragment (1-45) was not identified in either patient. In each case, the four exons of the TTR gene were of normal sequence. CONCLUSIONS: In familial amyloidosis resulting from deposition of TTR Met 30 (Swedish-type familial amyloidosis), full-length TTR molecules (some mutant) usually predominate, and TTR fragments lacking three of the eight beta-strands (nonmutant) form a major part of the fibril in senile systemic amyloidosis. This may indicate a difference in the fibrillogenesis between these two forms of TTR-derived amyloidosis. We propose that the name senile systemic amyloidosis be used only for normal-sequence TTR amyloidosis occurring in advanced age.

Detection of K-ras oncogene mutations in bronchoalveolar lavage fluid for lung cancer diagnosis.

BACKGROUND: Lung cancer is the leading cause of cancer deaths in the United States. A long-standing goal of cancer researchers has been to develop tests that would facilitate earlier diagnosis and treatment of lung cancer and thereby decrease mortality from this disease. Because cancer results from the accumulation of a variety of genetic events (e.g., mutations, rearrangements, and deletions) in genes controlling cell growth and differentiation, these changes might serve as diagnostically useful molecular markers. Activation of the K-ras oncogene by point mutations in codon 12, which occurs in many cases of lung adenocarcinoma, may serve as one such clinically useful molecular marker. For detection of K-ras point mutations in bronchoalveolar lavage fluid, in which small numbers of malignant cells are mixed with a population of predominantly genetically normal cells, the sensitivity of commonly used assays for ras mutations risks false-negative results. PURPOSE: By applying a highly sensitive assay, we investigated whether detection of K-ras codon 12 mutations in samples of bronchoalveolar lavage fluid could be clinically useful in diagnosing lung cancer. METHODS: We developed a highly sensitive assay for detecting K-ras codon 12 mutations based on an enriched polymerase chain reaction (PCR) technique. This technique was applied to 87 specimens of bronchoalveolar lavage fluid specimens that were obtained from 86 patients, and associated tumor biopsy specimens obtained from 35 of these patients who underwent diagnostic bronchoscopy for clinically suspected lung cancer. Statistical comparisons were performed by using the two-tailed Fisher's exact test [corrected]. RESULTS: Of 52 patients with confirmed lung cancer, samples of bronchoalveolar lavage fluid from 16 patients contained K-ras codon 12 mutations, including 14 (56%) of 25 patients with lung adenocarcinomas, one (33%) of three with bronchoalveolar carcinomas, one (20%) of five with large-cell carcinomas, and none of the 14 with squamous cell carcinomas. Mutations were detected in four additional cases in which cancer was suspected but had not been histologically confirmed. Tissue samples from 35 of the patients all yielded the identical K-ras codon 12 genotype found in the corresponding samples of bronchoalveolar lavage fluid. No mutation was found in any sample from 30 patients with diagnoses other than non-small-cell lung cancer. Thus, for those cases in which tissue was available and tested, the sensitivity and specificity of detecting K-ras mutations in bronchoalveolar lavage fluid for diagnosing K-ras mutation-positive lung cancer were both 100%. For nine patients, K-ras mutations were detected in bronchoalveolar lavage fluid obtained during otherwise nondiagnostic bronchoscopies. CONCLUSIONS: Our data demonstrate that sensitive detection of K-ras codon 12 mutations can serve as an important adjunct to cytology in the diagnosis of lung cancer. IMPLICATIONS: Detection of these mutations could lead to earlier cancer diagnosis and less need for invasive diagnostic procedures.

Variable-mode helical CT: imaging protocols.

Helical CT provides a rapidly acquired, accurately registered, two-dimensional data set during the phase of maximum vascular and parenchymal enhancement. The z-axis can be covered by using either a single acquisition, single breath-hold technique or by using multiple helical groups with intergroup delays for the patient's breathing. The latter approach, called variable-mode helical CT, allows large z-axis coverage of more than one anatomic region during injection of a single bolus of contrast material. We discuss helical scanning protocols, both single-acquisition and variable-mode, that we have developed for detection of disease involving major blood vessels (pulmonary arteries and aorta), perivascular tissue planes (neck, thorax, and pelvis), and abdominal viscera (liver, pancreas, and kidneys).

Increased prevalence of K-ras oncogene mutations in lung adenocarcinoma.

Reported estimates of ras mutation prevalence in lung adenocarcinoma of 15-24% may be underestimates because of the insensitivity of the assays used. We have devised a rapid, non-radioactive assay for ras mutations, which detects 1 mutant allele/10(3) normal alleles and have used it to study DNA isolated from 53 lung tumor samples (including 28 adenocarcinomas) previously analyzed by PCR/allele specific oligonucleotide hybridization, which is less sensitive. We detected mutations in 13 of 28 samples, including 7 not detected by PCR/allele specific oligonucleotide hybridization. We also found ras mutations in 14 of 25 previously unstudied samples (56%). Our results indicate that the prevalence of K-ras codon 12 mutations in lung adenocarcinoma is higher than previously reported; thus, ras mutations may be more clinically useful as molecular markers for lung cancer than has been appreciated.

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.

Molecular genetic tumor markers in the early diagnosis and screening of non-small-cell lung cancer.

BACKGROUND: Little progress has been made in decreasing lung cancer mortality by applying conventional methods to early diagnosis and screening. Recent advances in molecular oncology, however, have provided tools which may be of use in this area. Many genes involved in controlling cell growth and differentiation are abnormal in lung cancer cells. Such genes include K-ras, p53, rb, myc, her2/neu, and probably one or more tumor suppressor genes on chromosome 3p. The involvement of these genes in lung cancer is reviewed. The K-ras oncogene contains a mutation in codon 12 in many cases of non-small-cell lung cancer, particularly adenocarcinoma, and is thus a potentially useful lung cancer tumor marker. DESIGN; We have developed a highly sensitive, simple assay for ras mutations, and applied it to bronchoalveolar lavage fluid obtained from patients undergoing evaluation for suspected lung cancer. RESULTS: In many cases, the ras assay was more sensitive than routine cytology and histopathology, demonstrating that this is a potentially clinically useful assay. CONCLUSION: Molecular genetic tumor markers, including mutations in ras and other genes, and/or immunohistochemical tumor markers, may provide tools which can be applied to bronchoalveolar lavage fluid or sputum, for use in diagnostic tests and in screening programs. The use of such markers may lead to decreased lung cancer mortality.