Absence of linkage disequilibrium at amyloid precursor protein gene locus in Japanese familial Alzheimer's disease with 717Val-->Ile mutation

To date, eleven independent early onset familial Alzheimer's disease (EOFAD) pedigrees with the 717Val-->Ile mutation of amyloid precursor protein (APP) gene have been identified. Interestingly, five pedigrees have been of Japanese origin. The apparent ethnic prediction of this mutation raises the possibility that there is a founder effect in Japan. If the hypothesis holds true, we can expect the presence of linkage disequilibrium at the APP locus. We did not, however, observe any significant linkage disequilibrium at any locus of APP or the adjacent GT12 locus in the five Japanese EOFAD probands with the 717Val-->Ile mutation. The result indicates that a founder effect would probably not be present in Japanese EOFAD pedigrees with the 717Val-->Ile mutation.     

Enhanced vulnerability to apoptotic cell death in sporadic Alzheimer's disease

All important neuropathological features like neurodegeneration and amyloid deposition occur similarly in familial and in sporadic Alzheimer's disease (AD) patients, suggesting a common pathogenetic mechanism. We investigated whether defective vulnerabilty to the induction of the apoptotic cell death pathway might be one of the underlying mechanisms leading to progressive cell death in sporadic AD. Our test could prove useful in supporting the diagnosis of AD and in predicting individuals predisposed to AD.     

ApoE allele frequencies in Italian sporadic and familial Alzheimer's disease

Recent studies have provided evidence of association of apolipoprotein E (ApoE) epsilon 4 allele and late onset familial and sporadic Alzheimer's disease (AD). Epidemiological studies have established allelic variation at the ApoE locus. We have analyzed the ApoE gene polymorphism in a sample of 446 Italian subjects. Our data confirm a significant association between epsilon 4 allele and sporadic AD. The frequency of epsilon 4 allele in early onset familial AD patients was comparable to control values suggesting that epsilon 4 allele does not represent a risk factor for early onset familial AD (EOFAD). Moreover, we found a not previously reported association between ApoE epsilon 2 allele and sporadic AD and EOFAD.     

MEN1 gene mutation analysis of sporadic adrenocortical lesions

To clarify the role of the MEN1 gene in the tumorigenesis of sporadic adrenocortical tumors, we performed a molecular study on 35 adrenocortical lesions including 6 hyperplasias, 19 adenomas and 10 carcinomas. Loss of heterozygosity (LOH) of the MEN1 gene was assessed by PCR using an intragenic (D11S4946) and 2 flanking microsatellite markers (D11S4936, PYGM) and/or fluorescence in situ hybridization (FISH) with a 40-kb cosmid probe containing the MEN1 gene. The complete coding sequence of the MEN1 gene was screened for mutations using non-radioactive, PCR-based single-strand conformation polymorphism (SSCP) analysis and MDE heteroduplex gel electrophoresis. PCR-LOH and FISH analyses performed in 29 tumors (PCR-LOH in 4, FISH in 17 and both in 8 tumors) revealed allelic deletion of the MEN1 locus in 8 (27.5%) and at 11q13 in 9 (31%) tumors. Furthermore, the frequency of LOH at 11q13 was significantly higher in adrenocortical carcinomas (60%) than in benign lesions (11%). Mutation analysis of tumor samples revealed 9 polymorphisms in 7 tumors (S145S, R171Q, R171Q together with L432L) but no mutations, with the exception of one adrenocortical adenoma. The latter tumor contained a somatic E109X stop codon mutation in exon 2 and a 5178-9G-->A splice mutation in intron 4, which was also detectable in various nontumorous tissues and blood indicative of a germ-line mutation. The patient, who had no clinical signs or family history of MEN1, later also developed a neuroendocrine carcinoma (atypical carcinoid) of the lung. Our findings indicate that inactivating mutations of the MEN1 tumor-suppressor gene appear not to play a prominent role in the development of sporadic hyperplastic or neoplastic lesions of the adrenal cortex and that the newly reported 5178-9G-->A splice mutation in intron 4 might cause a variant of the MEN1 phenotype.     

Brain insulin and insulin receptors in aging and sporadic Alzheimer's disease

The search for the causes of neurodegenerative disorders is a major theme in brain research. Acquired disturbances of several aspects of cellular metabolism appear pathologically important in sporadic Alzheimer's disease (SDAT). Among these brain glucose utilisation is reduced in the early stages of the disease and the regulatory enzymes important for glucose metabolism are reduced. In the brain, insulin, insulin-like growth factors and their receptors regulate glucose metabolism and promote neuronal growth. To detect changes in the functional activity of the brain insulin neuromodulatory system of SDAT patients, we determined the concentrations of insulin and c-peptide as well as insulin receptor binding and IGF-I receptor binding in several regions of postmortem brain cortex during aging and Alzheimer's disease. Additionally, we performed immunohistochemical staining with antibodies against insulin in neocortical brain areas in SDAT and controls. We show for the first time that insulin and c-peptide concentration in the brain are correlated and decrease with aging, as do brain insulin receptor densities. Weak insulin-immunoreactivity could be demonstrated histochemically in pyramidal neurons of controls, whereas in SDAT a stronger insulin-immunoreactivity was found. On a biochemical level, insulin and c-peptide levels were reduced compared to middle-aged controls, but were unchanged compared to age-matched controls. Brain insulin receptor densities in SDAT were decreased compared to middle-aged controls, but increased in comparison to age-matched controls. IGF-I receptor densities were unchanged in aging and in SDAT. Tyrosine kinase activity, a signal transduction mechanism common to both receptor systems, was reduced in SDAT in comparison to middle-aged and age-matched control groups. These data are consistent with a neurotrophic role of insulin in the human brain and a disturbance of insulin signal transduction in SDAT brain and favor the hypothesis that insulin dependent functions may be of pathogenetic relevance in sporadic SDAT.     

The Glu318Gly mutation of the presenilin-1 gene does not necessarily cause Alzheimer's disease

In early-onset familial Alzheimer's disease (AD) pathogenic mutations have been found in the amyloid precursor protein (APP) gene and in the presenilin (PS)-1 and PS-2 genes. We screened for mutations in these genes in 20 patients with familial AD from the Finnish population. In addition, we sampled 41 sporadic AD patients and 59 controls to test for mutations identified in our familial AD cases. We detected an A-to-G transition in the PS-1 gene, resulting in a glutamic acid (Glu)-to-glycine (Gly) substitution at codon 318 in 2 familial and 2 sporadic AD patients. The Glu318Gly mutation has previously been reported to cause AD. We also found the Glu318Gly mutation in 4 healthy aged controls (range, 74-87 years). We thus conclude that the mutation is most likely a rare polymorphism not related to AD.     

Japanese siblings with missense mutation (717Val --> Ile) in amyloid precursor protein of early-onset Alzheimer's disease

We report Japanese siblings with the missense mutation 717Val --> Ile in the amyloid precursor protein. The maternal grandmother died of an unknown dementing disorder. The proband's mother had gradually increasing amnesia beginning at age 64, which was diagnosed as Alzheimer's disease (AD). She died in a psychiatric hospital (duration of illness: 16 years). Both the proband and her elder sister were affected at about age 55 years. Disturbances of memory, judgment, and emotion, as well as personality changes, occurred first, with dementia eventually predominating. The elder sister died of pneumonia (duration of illness: 9 years). The amyloid precursor protein (APP) gene was analyzed from each sibling. Genomic DNAs obtained from blood samples were amplified by the polymerase chain reaction (PCR) method. PCR products were digested with the restriction enzyme Bcl I. The resulting restriction fragment length polymorphisms (RFLPs) showed the missense mutation 717Val --> Ile in both patients, but not in a normal control. DNA sequencing showed the presence of the 2149G --> A missense mutation only in the patients. We conclude that this familial AD may originate from the missense mutation 717Val --> Ile in the amyloid precursor protein gene and that the clinical picture is typical of AD, except for normal-pressure hydrocephalus and psychiatric phenomena.     

Amyloid precursor protein (APP) in the striatum in Alzheimer's disease: an immunohistochemical study

Increasing recognition of diffuse plaques has raised questions about the differences between diffuse and neuritic plaques, particularly in regard to the role of amyloid precursor protein (APP) processing in their formation. To address this issue, corpus striatum (containing almost exclusively diffuse plaques) and cerebral cortex (containing an admixture of plaque types) from patients with Alzheimer's disease (AD) were examined immunohistochemically with antibodies to domain-specific sites of APP (N-terminal, C-terminal, beta A4-related, isoform-specific, and other epitopes). Striatal plaques labeled strongly with beta A4 antibodies as did cortical plaques in AD and the occasional diffuse plaques in cortex from nondemented elderly controls. Weak labeling of some cortical neuritic plaques but not diffuse plaques was observed with antibodies directed against other APP epitopes. Electron microscopy of diffuse plaque-rich striatum in AD cases revealed only rare degenerating neurites without apparent fibrillar amyloid; no changes were noted in the plaque-free striatum of controls. These results suggest that antibodies to beta A4 recognize not only fibrillar amyloid of neuritic plaques but also antigenic determinants of diffuse plaques which lack fibrillar amyloid. Furthermore, the finding that antibodies to non-A4 domains of APP labeled only cortical but not striatal plaques suggests that APP processing mechanisms in cortical and striatal tissues may differ.     

Mitochondrial ribosomal RNA gene mutation in a patient with sporadic aminoglycoside ototoxicity

PURPOSE: Aminoglycoside-induced deafness has been described in a number of Chinese pedigrees. In nearly all of these families, affected individuals were related through the maternal side. Because mitochondrial DNA is transmitted exclusively through mothers, it had been speculated that a mutation in the mitochondrial DNA might predispose these maternally related family members to aminoglycoside ototoxicity. Recently, we analyzed three such families with multiple cases of ototoxic deafness and identified a pathogenic mutation in the mitochondrial 12S ribosomal RNA gene at nucleotide position 1555. The purpose of the current study is to analyze individuals with no family history of deafness, who had severe hearing loss after aminoglycoside exposure, for presence or absence of this particular mitochondrial DNA mutation. MATERIALS AND METHODS: Blood was obtained from 36 Chinese individuals who became deaf after aminoglycoside exposure and had no family history of deafness. The DNA of these individuals was extracted, amplified by the polymerase chain reaction, and analyzed for the mitochondrial ribosomal RNA gene mutation by allele-specific oligonucleotide hybridization and Southern blot analysis. RESULTS: In one of these 36 sporadic cases, we identified the nucleotide 1555 A-->G mutation in the mitochondrial genome. CONCLUSION: This finding implies that a small proportion of individuals at risk for aminoglycoside ototoxicity harbor the specific mitochondrial DNA mutation identified in the familial cases. In these individuals, a genetic susceptibility to the ototoxic effects of aminoglycosides can be diagnosed, and deafness can be prevented in maternal relatives by avoiding the use of these antibiotics.     

Loricrin gene mutation in a Japanese patient of Vohwinkel's syndrome

It has been suggested that nitric oxide (NO) could be implicated in the pathogenesis of multiple sclerosis (MS). Recently, two groups reported increased cerebrospinal fluid (CSF) nitrate levels (oxidation product that provides an indirect estimation of NO) in MS patients. However, another group did not confirm these findings. We studied the CSF and plasma levels of nitrate with a kinetic cadmium reduction method in 11 MS patients and 25 matched controls. The CSF nitrate levels and the CSF/plasma nitrate ratio did not differ significantly between the two study groups. Plasma nitrate levels were nearly significantly lower in MS patients. CSF and plasma nitrate levels did not correlate with age at onset and duration of the disease in the patient group. These data suggest that measurement of CSF levels of nitrate is not a marker of the activity of MS.     

A novel point mutation in congenital erythropoietic porphyria in two members of Japanese family

The molecular basis of the uroporphyrinogen III synthase (UROIIIS) deficiency was investigated in two members of a Japanese family. This defect in heme biosynthesis is responsible for a rare autosomal recessive disease: congenital erythropoietic porphyria (CEP) or Gnther's disease. The first patient was homoallelic for a novel missense mutation: a T to C transition of nucleotide 634 that predicted a serine to proline substitution at residue 212 (S212P). The second patient appeared heteroallelic, carrying the same missense mutation and a nonsense mutation: a C to T change at nucleotide 745, resulting in a premature stop at codon 249, instead of a glutamine (Q249X). The corresponding mutated proteins were expressed in Escherichia coli and no residual activity was observed. A family study was also performed to determine the carrier status.     

Lack of mutation in the WAF1/CIP1 gene during bovine leukemia virus-induced leukemogenesis

As a cyclin-dependent kinases (Cdks) inhibitor (CDI), the protein p21WAF1/CIP1 is able to interfere with cell cycle progression. Its expression is upregulated by wild-type p53, and the p21WAF1/CIP1 protein appears to be a potent effector of the p53-dependent cell cycle regulatory pathway. We have previously reported that p53 mutations frequently occur during bovine leukemia virus (BLV)-induced leukemogenesis in cattle but not in sheep. Therefore, we have investigated the involvement of p21WAF1/CIP1 mutations in the tumorigenic process associated with BLV. We first cloned the bovine and ovine WAF1 genes and determined the complete nucleotide sequences of their second coding exons. These sequences share respectively 79% and 80% homology with those of the human counterpart exon. In order to screen for mutations that could be associated with BLV-induced pathogenicity, we performed single strand conformation polymorphism (SSCP) assays on the WAF1 genes from BLV-induced tumors. No WAF1 mutations were detected in any of the ten BLV-induced bovine tumor samples. Among eleven sheep tumors and three ovine cell lines, only one sample revealed a single mutation in the WAF1 coding sequence, but this mutation was silent at the translational level. We concluded that mutations of the WAF1 gene are not involved in the development of the tumors during BLV-induced leukemogenesis.     

Single somatic ras gene point mutation in soft tissue malignant fibrous histiocytomas

The frequency of ras gene mutations in human soft tissue malignant fibrous histiocytomas within and around the hot spot codons (12, 13, and 61) of all ras genes, (H-ras-1, K-ras-2, and N-ras) was studied by nested polymerase chain reaction and direct DNA sequencing from archival formalin-fixed, paraffin-embedded tissue. Light microscopy and immunohistochemistry served to define malignant fibrous histiocytoma. All of the four differentiation subtypes (storiform-pleomorphic, inflammatory, myxoid, and giant cell) were investigated. Nine of thirty-two malignant fibrous histiocytomas (28%) contained ras gene point mutations. The highest incidence was found in the myxoid subtype (four of nine). H-ras-1 gene codon 12.2 was the only codon affected and contained in all mutated cases a GGC-->GTC exchange. Seven of the nine mutations were homozygous and probably affected more than 80% of the tumor DNA. The flanking regions of all hotspot codons did not contain any point mutation. The presence of a single and often homozygous point mutation of the H-ras-1 gene, especially in myxoid malignant fibrous histiocytoma could serve as a basis for further genomic discrimination of myxoid sarcomas.     

Cellular UDP-glucose deficiency caused by a single point mutation in the UDP-glucose pyrophosphorylase gene

We previously isolated a mutant cell that is the only mammalian cell reported to have a persistently low level of UDP-glucose. In this work we obtained a spontaneous revertant whose UDP-glucose level lies between those found in the wild type and the mutant cell. The activity of UDP-glucose pyrophosphorylase (UDPG:PP), the enzyme that catalyzes the formation of UDP-glucose, was in the mutant 4% and in the revertant 56% of the activity found in the wild type cell. Sequence analysis of UDPG: PP cDNAs from the mutant cell showed one missense mutation, which changes amino acid residue 115 from glycine to aspartic acid. The substituted glycine is located within the largest stretch of strictly conserved residues among eukaryotic UDPG:PPs. The analysis of the cDNAs from the revertant cell indicated the presence of an equimolar mixture of the wild type and the mutated mRNAs, suggesting that the mutation has reverted in only one of the alleles. In summary, we demonstrate that the G115D substitution in the Chinese hamster UDPG:PP dramatically impairs its enzymatic activity, thereby causing cellular UDP-glucose deficiency.     

Molecular analysis of the RET proto-oncogene in patients with sporadic medullary thyroid carcinoma: a novel point mutation in the extracellular cysteine-rich domain

A filiform stenosis of the popliteal artery was examined by sonography and the reason for the cystic lesion in the arterial wall was found to be an uncommon type of cystic degeneration of the adventitia. The narrow lumen could be demonstrated by sonography and the lesion was punctured under ultrasound control. A viscous secretion was removed and the patient's claudication was cured without recourse to surgery.     

A point mutation in the gene encoding the Rubisco large subunit interferes with holoenzyme assembly

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), a key enzyme of photosynthetic CO2 fixation, is composed of 8 large and 8 small subunits. The Rubisco-deficient Nicotiana tabacum mutant Sp25 is able to synthesize the peptides for both subunits but does not contain any active holoenzyme. The phenotype is maternally inherited and thus caused by a mutation in the chloroplast genome, which also encodes the Rubisco large subunit. A comparison of the nucleotide sequences of the large subunit gene of the Sp25 mutant with that of the wild-type tobacco revealed a single nucleotide change in the Sp25 mutant. This resulted in an amino acid substitution at Gly-322, which was replaced by serine.