Anthony J. Brookes

Apolipoprotein E ϵ4 allele frequency in patients with Lewy body dementia, Alzheimer's disease and age-matched controls

Genotypes of apolipoprotein E (ApoE) were determined by polymerase-chain reaction (PCR) amplification and restriction-sizing of brain DNA from 39 cases of Lewy body dementia (LBD), 68 senile Alzheimers disease (AD) patients and 47 neuropathologically validated non-demented controls. There was a 3-fold increase in the epsilon 4 allele frequency in both LBD and AD groups compared with controls. These results indicate that LBD and AD share the epsilon 4 allele of ApoE as a major risk factor for the development of disease and suggest a similarity in disease aetiology.

Synuclein proteins and Alzheimer's disease

In Alzheimers disease, synuclein/NAC (non-amyloid beta component of Alzheimers disease amyloid) proteins are found in presynaptic cholinergic nerve terminals that degenerate early in Alzheimers disease, and they are also found closely linked to beta-amyloid fibrils in senile plaques. Synuclein/NAC proteins provide a potential molecular link between the degeneration of cholinergic nerve terminals, and the formation of plaques, and might have a primary role in their development.

High-resolution mapping of SNCA encoding α-synuclein, the non-Aβ component of Alzheimer’s disease amyloid precursor, to human chromosome 4q21.3→q22 by fluorescence in situ hybridization

The human α-synuclein gene (SNCA) was previously identified as the non-Aβ component of Alzheimer’s disease amyloid precursor (NACP). A cosmid clone containing this gene has been isolated and mapped by

The NIK protein kinase and C17orf1 genes: chromosomal mapping, gene structures and mutational screening in frontotemporal dementia and parkinsonism linked to chromosome 17

Abstract Full exon-intron structures are presented for the NIK serine/threonine protein kinase gene and a novel gene termed C17orf1. By in situ hybridisation and radiation hybrid mapping, a cosmid (cDD-Z) that contains regions of both of these genes has been localised between markers D17S800 and D17S791 at chromosome 17q21. The two genes are thus positional candidates for the mutant locus underlying frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), a disease for which NIK is also a good biological candidate. Using exon-intron maps, a genomic DNA sequencing based mutation screen has been performed for the NIK and C17orf1 genes in a chromosome 17-linked FTDP-17 pedigree. Two silent single-base variations were detected in C17orf1. No alterations were restricted to DNA samples from patients, thus excluding the C17orf1 and NIK genes as likely sites of mutation FTDP-17.

Novel transcribed sequences neighbouring a translocation breakpoint associated with schizophrenia

A 1.3Mb chromosome 11-specific yeast artificial chromosome (YAC) that spans a t(1;11) translocation breakpoint associated with major psychosis has been used to enrich cDNAs that are encoded within it and expressed in the human foetal brain. Database analysis of the selected fragments led to the identification of 54 clones matching alpha-tubulin, 4 fragments matching two anonymous human expressed sequence tags (ESTs) and 8 fragments giving no database matches. The clones matching alpha-tubulin led to the identification of a novel alpha-tubulin locus located approximately 250 kb proximal to the translocation breakpoint. Extensive sequence and expression analysis of this locus suggests that this is a processed pseudogene, although a long open reading frame is maintained and the possibility that an abnormally acting protein may be expressed in a highly tissue or developmental specific manner cannot be discounted. The novel cDNA fragments map up to 700 kb proximal to the translocation breakpoint and are associated with potential CpG islands. Reverse transcriptase polymerase chain reaction (RT-PCR) expression analysis and high resolution genomic mapping suggest that they may comprise up to three novel genes. No major disruption of the identified fragments could be detected in the genomic DNA of translocation carriers. The psychosis associated with this translocation may therefore be due to position effects on the transcription of these genes or an involvement of translocated chromosome 1 sequences.

Alu-based vectorettes and splinkerettes: More efficient and comprehensive polymerase chain reaction amplification of human DNA from complex sources

Alu-polymerase chain reaction (PCR) is widely used to amplify human specific fragments from complex heterologous DNAs, such as somatic cell hybrids or yeast artificial chromosome (YAC) recombinants, but the fragments amplified are limited in number and are nonrepresentative. This report describes a modified one-sided alu-PCR technique, which offers better representation of amplified sequences while maintaining human specificity. The method relies on the ligation of partially mismatched double-stranded oligonucleotides (vectorettes or splinkerettes) to endonuclease-restricted DNA and universal priming with a single alu-consensus primer, the complement to which is the unpaired region. Alu-vectorette and alu-splinkerette-PCR of two somatic cell hybrids results in a greater complexity of products than alu-PCR alone. The advantage of alu-splinkerette over alu-vectorette-PCR is the elimination of nonspecific priming owing to the presence of the vectorette primer and to an increase in the product size range, a consequence of the difference in the splinkerette design. Alu-splinkerette-PCR is a useful technique for generating new and more comprehensive markers of the human sequences contained in somatic cell hybrids and YACs.

Novel transcribed sequences represented in the complex genomic region 5q13

YACs from the complex repetitive human genomic region 5q13, spanning the spinal muscular atrophy (SMA) locus, have been searched for transcribed sequences using the method of End Ligation Coincident Sequence Cloning. Six transcripts (PT1-6) have been identified, three of which (PT4, PT5 and PT6) are novel. Five of these elements hybridise to multiple loci in 5q13, but PT5 is single copy and maps very close to markers that show linkage disequilibrium with SMA.

Identifying genes within microdissected genomic DNA: Isolation of brain expressed genes from a translocation region associated with inherited mental illness

An improved protocol has been developed for physical enrichment of cDNA sequences by hybridization to genomic DNA. When applied to microdissection recombinants derived from a translocation breakpoint region associated with inherited mental illness, a single cycle of the procedure permitted enriched cDNAs to be visualized directly by agarose gel electrophoresis. Hybridization screening of a library of clones derived from the enriched cDNAs, employing the genomic resource as a probe, led to the identification of six novel gene fragments. This general approach to the isolation of regionally encoded genes could be applied to any subchromosomal interval as a first step towards global transcription map construction.

Coincident sequence cloning : a new approach to genome analysis

Many analytical molecular genetic techniques developed over the past decade have evolved primarily to tackle the problem of targeting or isolating sequences of interest in complex mixtures. A new approach to this problem, Coincident Sequence Cloning (CSC), involves integrating a pair of DNA mixtures in such a way as to isolate any shared sequence components.