Transcription of Alu DNA elements in blood cells of sporadic Creutzfeldt-Jakob disease (sCJD)

Abstract

Alu DNA elements were long considered to be of no biological significance and thus have been only poorly defined. However, in the past Alu DNA elements with well-defined nucleotide sequences have been suspected to contribute to disease, but the role of Alu DNA element transcripts has rarely been investigated. For the first time, we determined in a real-time approach Alu DNA element transcription in buffy coat cells isolated from the blood of humans suffering from sporadic Creutzfeldt-Jakob disease (sCJD) and other neurodegenerative disorders. The reverse transcribed Alu transcripts were amplified and their cDNA sequences were aligned to genomic regions best fitted to database genomic Alu DNA element sequences deposited in the UCSC and NCBI data bases. Our cloned Alu RNA/cDNA sequences were widely distributed in the human genome and preferably belonged to the “young” Alu Y family. We also observed that some RNA/cDNA clones could be aligned to several chromosomes because of the same degree of identity and score to resident genomic Alu DNA elements. These elements, called paralogues, have purportedly been recently generated by retrotransposition. Along with cases of sCJD we also included cases of dementia and Alzheimer’s disease (AD). Each group revealed a divergent pattern of transcribed Alu elements. Chromosome 2 was the most preferred site in sCJD cases, besides chromosome 17; in AD cases chromosome 11 was overrepresented whereas chromosomes 2, 3 and 17 were preferred active Alu loci in controls. Chromosomes 2, 12 and 17 gave rise to Alu transcripts in dementia cases. The detection of putative Alu paralogues widely differed depending on the disease. A detailed data search revealed that some cloned Alu transcripts originated from RNA polymerase III transcription since the genomic sites of their Alu elements were found between genes. Other Alu DNA elements could be located close to or within coding regions of genes. In general, our observations suggest that identification and genomic localization of active Alu DNA elements could be further developed as a surrogate marker for differential gene expression in disease. A sufficient number of cases are necessary for statistical significance before Alu DNA elements can be considered useful to differentiate neurodegenerative diseases from controls.

Acknowledgements

Financial support was given by the DFG (ZI 568/3-2 and KA 864/2-1) and by the German Primate Center (DPZ). This study was funded by Robert Koch-Institut through funds of the Federal Ministry of Health (grant no 1369-341 to I.Z.).

Figures and Tables

Figure 1 Experimental approach to identify transcribed “active” Alu DNA elements and their chromosomal location. A stringent protocol was developed to isolate Alu RNA and to prepare cDNA sequences in order to search of the active, i.e., expressed/transcribed Alu DNA elements within the human genome.

Figure 2 Cloning of Alu RNA/cDNA and its sequence for alignment. The amplicon was 277 bps in size and the sequence for alignment 257 bps in size. The Eco RI cloning sites and the primer sites are in bold and the 5′ and 3′ sites for alignments are underlined. The clone shown: sequence 884 clone no. 1 sCJD and sequence obtained with M13 reverse primer.

Figure 3 Frequency of active Alu DNA elements on chromosomes of healthy and diseased humans. The numbers of RNA/cDNA clones targeting individual chromosomes are given. Clearly, chromosome 2 is the chromosome where transcribed = active Alu DNA elements preferentially reside.

Figure 4 Frequency of Alu Y transcripts in buffy coat cells of healthy humans and cases with neurodegenerative diseases. Numbers of Alu Y RNA/cDNA clones obtained from healthy controls, dementia and Alzheimer disease cases are shown in (A) and sporadic CJD are shown in (B). The prion protein genotypes are either homozygous or heterozygous for methionine or valine: M/M, V/V or M/V.

Figure 5 Intergenic and intragenic locations of active Alu DNA elements. A detailed computer search allowed an exact determination of the genomic location where the transcribed Alu DNA element was located, i.e., within genes or flanking genes: intragenic and splice sites or between genes: intergenic.