Study of the Alzheimer's A4 precursor gene promoter region by genomic sequencing using Taq polymerase

The beta-amyloid protein has been identified as the prominent component of the fibrillary aggregates of the neuritic plaques found in Alzheimer's disease (AD). In this paper, the DNA methylation pattern of the promoter region of the Alzheimer's disease amyloid precursor gene (PAD) was assessed using the recently developed genomic sequencing technique with Taq polymerase. We analyzed seven potential methylation sites between position -460 and -275 of the PAD promoter. Three of the CpG dinucleotides we analyzed are located in the flanking regions of the AP-1 binding site and heat-shock response element consensus sequences. Of the seven CpG dinucleotides present in this region, we found none to be methylated. This finding indicates that, in healthy brain tissue, cytosine methylation of this binding motif seems not to affect protein/DNA interaction. However, it remains to be determined whether methylation of these sites is significant in AD patients.     

The PreA4(695) precursor protein of Alzheimer''s disease A4 amyloid is encoded by 16 exons.

Alzheimer's disease (AD) is characterized by the cerebral deposition of fibrillar aggregates of the amyloid A4 protein. Complementary DNA's coding for the precursor of the amyloid A4 protein have been described. In order to identify the structure of the precursor gene relevant clones from several human genomic libraries were isolated. Sequence analysis of the various clones revealed 16 exons to encode the 695 residue precursor protein (PreA4(695] of Alzheimer's disease amyloid A4 protein. The DNA sequence coding for the amyloid A4 protein is interrupted by an intron. This finding supports the idea that amyloid A4 protein arises by incomplete proteolysis of a larger precursor, and not by aberrant splicing.     

Identification and differential expression of a novel alternative splice isoform of the beta A4 amyloid precursor protein (APP) mRNA in leukocytes and brain microglial cells.

The gene for the beta A4-amyloid precursor protein (APP) consists of 19 exons which code for a typical N- and O-glycosylated transmembrane protein with four extracellular domains followed by the transmembrane domain and a short cytoplasmic domain. The beta A4-amyloid sequence is part of exons 16 and 17. Several APP isoforms can be generated by alternative splicing of exons 7 and 8, encoding domains with homologies to Kunitz-type protease inhibitors and the MRC OX-2 antigen, respectively. The mechanism by which the pathological beta A4 is generated is unknown, it is however a critical event in Alzheimer's disease and is distinct from the normally occurring cleavage and secretion of APPs within the beta A4 sequence. We report here for the first time considerable APP mRNA expression by rat brain microglial cells. In addition we showed by S1 nuclease protection and polymerase chain reaction analysis of reverse transcribed RNA (RT-PCR) that T-lymphocytes, macrophages, and microglial cells expressed a new APP isoform by selection of a novel alternative splice site and exclusion of exon 15 of the APP gene. This leads to a transmembrane, beta A4 sequence containing APP variant, lacking 18 amino acid residues close to the amyloidogenic region. The use of this novel alternative splice site alters the structure of APP in close proximity to the beta A4 region and thus may determine a variant, potentially pathogenic processing of leukocyte-derived APP in brain.     

Frequency of abnormal human haemoglobins caused by C----T transitions in CpG dinucleotides

A large part of human genetic disease apparently arises from deamination of cytosine residues in methylated CpG dinucleotides. Their mutation rate is known to be high when C is present as 5-methyl-cytosine, but is believed to be normal when it is unmethylated. The beta-globin gene contains five, the gamma-globin gene two, and each of the alpha-globin genes contains 35 CpG dinucleotides. The CpG dinucleotides in the beta and gamma-globin genes are methylated, while those in the alpha-globin genes are under-methylated. One would therefore have expected the CpG dinucleotides to be a frequent source of mutations in the beta and gamma-globin genes, but not in the alpha-globin genes. In fact, the evidence points to CpG dinucleotides being a frequent source of mutations in both the alpha and beta-globin genes. This suggests either that the mutation rates of both methylated and unmethylated CpG dinucleotides are abnormally high, which conflicts with published evidence, or that there is a finite chance of some of these in the alpha-globin genes of certain individuals being methylated and therefore subject to mutation.     

The CpG island in the 5' region of the G6PD gene of man and mouse

The nucleotide (nt) sequence of the entire CpG island in the 5' region of the human glucose-6-phosphate dehydrogenase-encoding gene (G6PD) and of the corresponding region in mouse was determined. In comparison to the human gene, the 5' region of the mouse G6PD gene has highly reduced G + C and CpG dinucleotide content, but maintains the functional features of a CpG island, as it is differentially methylated on the active vs. the inactive X chromosome. In addition to the expected conservation of exons, nt sequence comparison showed that several boxes are highly conserved between the two species in the 5' flanking DNA and in the first intron. Moreover, the conservation of the position of most CpG dinucleotides in the promoter region and in one of the upstream boxes, at about -900, gives support to the hypothesis that, in each island, specific CpGs play a major role in the regulation of gene expression.     

Alzheimer's disease amyloid peptide is encoded by two exons and shows similarity to soybean trypsin inhibitor

To better understand the processing of the Alzheimer disease amyloid precursor protein, we have cloned and sequenced that region of the human genome coding for the amyloid peptide. Two exons separated by a 6.2kb intron define this region. Characterization of the A4 peptide amino acid sequence shows similarity to the structure of soybean trypsin inhibitor (Kunitz). Our observation describes a different region of PreA4 than the previously characterized domain of larger amyloid precursor molecules PreA4 751 and 770(2). Moreover, the exon organization, Kunitz domain duplication and transmembrane location of A4 suggest that PreA4 is similar to growth factor precursors and thus may be processed similarly.     

Detection of amyloid beta protein precursor immunoreactivity in normal and Alzheimer's disease cerebrospinal fluid

The amyloid A4 (or beta protein), a 4.2 kD polypeptide, is a major component of amyloid deposits in the brains of patients with Alzheimer's Disease (AD). The self-aggregating amyloid A4 protein of AD is encoded as part of three larger proteins by the amyloid A4 precursor gene. The corresponding proteins have 695, 751 and 770 amino acid residues. To investigate the utility of amyloid beta protein precursor (A beta PP) as a diagnostic marker for AD an antiserum against a synthetic peptide (175-186), predicted from cDNA sequence for A beta PP, was used. The immunoreactivity of A beta PP in normal and AD cerebrospinal fluid (CSF) was measured by Western blot and detected with radiolabeled protein A. A total of fifty-seven CSF samples (AD = 27 and normal = 30) were analyzed for A beta PP immunoreactivity. A polyclonal antibody detected two major protein bands with apparent molecular weights of 105kD and 90kD both in normal and AD CSF. The difference between normal and AD CSF was not significant. These results indicate that immunoreactivity of A beta PP is present both in normal and AD CSF, and that the difference is too small to be used as a diagnostic marker.     

A homologous domain between the amyloid protein of Alzheimer's disease and the neurofilament subunits

Polypeptide (A4), which is derived from a larger precursor membrane protein (beta APP), is an important component of brain amyloid in Alzheimer's disease. The physiological function and the processing of this precursor are largely unknown. In order to elucidate the actual role of beta APP, we searched for domain homology with other proteins. The present study reveals the presence of a highly conserved region between the amyloid precursor and the 3 neurofilament subunits. Identical amino acids are present at about 45% of the positions aligned between the 4 sequences. These observations are discussed in terms of a possible involvement of positive ions in the maturation of these proteins and are in favour the implication of aluminium in Alzheimer's disease.     

Alzheimer's disease and control brain contain soluble derivatives of the amyloid protein precursor that end within the beta amyloid protein region.

The 39-43 amino acid beta amyloid protein (A beta) that deposits as amyloid in the brains of patients with Alzheimer's disease (AD) is encoded as an internal sequence within a larger membrane-associated protein known as the amyloid protein precursor (APP). In cultured cells, the APP is normally cleaved within the A beta to generate a large secreted derivative and a small membrane-associated fragment. Neither of these derivatives can produce amyloid because neither contains the entire A beta. Our study was designed to determine whether the soluble APP derivatives in human brain end within the A beta as described in cell culture or whether AD brain produces potentially amyloidogenic soluble derivatives that contain the entire A beta. We find that both AD and control brain contain nonamyloidogenic soluble derivatives that end at position 15 of the A beta. We have been unable to detect any soluble derivatives that contain the entire A beta in either the AD or control brain.     

LDLR Database (second edition): new additions to the database and the software, and results of the first molecular analysis.

Mutations in the LDL receptor gene (LDLR) cause familial hypercholesterolemia (FH), a common autosomal dominant disorder. The LDLR database is a computerized tool that has been developed to provide tools to analyse the numerous mutations that have been identified in the LDLR gene. The second version of the LDLR database contains 140 new entries and the software has been modified to accommodate four new routines. The analysis of the updated data (350 mutations) gives the following informations: (i) 63% of the mutations are missense, and only 20% occur in CpG dinucleotides; (ii) although the mutations are widely distributed throughout the gene, there is an excess of mutations in exons 4 and 9, and a deficit in exons 13 and 15; (iii) the analysis of the distribution of mutations located within the ligand-binding domain shows that 74% of the mutations in this domain affect a conserved amino-acid, and that they are mostly confined in the C-terminal region of the repeats. Conversely, the same analysis in the EGF-like domain shows that 64% of the mutations in this domain affect a non-conserved amino-acid, and, that they are mostly confined in the N-terminal half of the repeats. The database is now accessible on the World Wide Web at http://www.umd.necker.fr     

Secretory processing of the Alzheimer amyloid beta/A4 protein precursor is increased by protein phosphorylation.

The 39-43 residue polypeptide (amyloid beta protein, beta A4) deposited as amyloid in Alzheimer's disease (AD) is derived from a set of 695-770 residue precursors referred to as the amyloid beta A4 protein precursor (beta APP). In each of the 695, 751, and 770 residue precursors, the 43 residue beta A4 is an internal peptide that begins 99 residues from the COOH-terminus of the beta APP. Each holoform is normally cleaved within the beta A4 to produce a large secreted derivative as well as a small membrane associated fragment. Neither of these derivatives can produce amyloid because neither contains the entire beta A4 peptide. In this study, we employ cells stably transfected with full length beta APP695, beta APP751, or beta APP770 expression constructs to show that phorbol ester activation of protein kinase C substantially increases the production of secreted forms from each isoform. By increasing processing of beta APP in the secretory pathway, PKC phosphorylation may help to prevent amyloid deposition.     

Conserved regulatory elements in the promoter region of the N-CAM gene.

Genomic clones containing 5'-flanking sequences, the first exon, and the entire first intron from the chicken N-CAM gene were characterized by restriction mapping and DNA sequencing. A > 600-bp segment that includes the first exon is very G + C-rich and contains a large proportion of CpG dinucleotides, suggesting that it represents a CpG island. SP-1 and AP-1 consensus elements are present, but no TATA- or CCAAT-like elements were found within 300 bp upstream of the first exon. Comparison of the chicken promoter region sequence with similar regions of the human, rat, and mouse N-CAM genes revealed that some potential regulatory elements including a "purine box" seen in mouse and rat N-CAM genes, one of two homeodomain binding regions seen in mammalian N-CAM genes, and several potential SP-1 sites are not conserved within this region. In contrast, high CpG content, a homeodomain binding sequence, an SP-1 element, an octomer element, and an AP-1 element are conserved in all four genes. The first intron of the chicken gene is 38 kb, substantially smaller than the corresponding intron from mammalian N-CAM genes. Together with previous studies, this work completes the cloning of the chicken N-CAM gene, which contains at least 26 exons distributed over 85 kb.