Anita Wehnert

Apolipoprotein E genotype, atherosclerosis, and cognitive decline: the Rotterdam study

The apolipoprotein E4 allele (APOE epsilon 4) and atherosclerosis are risk factors for cognitive decline. We investigated whether the effects of APOE epsilon 4 and atherosclerosis on cognitive decline are independent. A population-based follow-up study was performed on 838 subjects who were non-demented at baseline. The Mini Mental State Examination (MMSE) score at follow-up was studied as a function of APOE epsilon 4 and atherosclerosis. Mild, non-significant effects on the MMSE score were found for atherosclerosis in the absence of APOE epsilon 4 and for APOE epsilon 4 in the absence of atherosclerosis. APOE epsilon 4 carriers with two or more indicators of atherosclerosis positive, had a significantly lower MMSE score at follow-up (mean difference -0.7 points; 95% confidence interval -1.1 to -0.2) relative to non-APOE epsilon 4 carriers with no evidence of atherosclerosis. Our findings suggest that the consequences of APOE epsilon 4 and atherosclerosis are not independent, and that particularly APOE epsilon 4 carriers with atherosclerosis are at increased risk of cognitive decline.

The Duffy blood group is linked to the α-spectrin locus in a large pedigree with autosomal dominant inheritance of Charcot-marie-Tooth disease type 1

SummaryThe α-spectrin locus (SPTA) on chromsome 1 maps to 1q22–q25 and α-spectrin specific probes detect restriction fragment length polymorphisms (RFLPs) with the endonucleases MspI and PvuII. The Duffy blood group (FY) has been mapped to the 1p21–q23 region. We found positive linkage between the α-spectrin and the Duffy loci with a maximal Lod score of 3.81 at θ=0.0 using the computer program MLINK. This indicates that both loci are very closely linked and probably localized to 1q22–q23.

Mutation analysis of the chromosome 14q24.3 dihydrolipoyl succinyltransferase (DLST) gene in patients with early-onset Alzheimer disease.

Linkage analysis studies have indicated that the chromosome band 14q24.3 harbours a major gene for familial early-onset Alzheimers disease (AD). Recently we localized the chromosome 14 AD gene (AD3) in the 6.4 cM interval between the markers D14S289 and D14S61. We mapped the gene encoding dihydrolipoyl succinyltransferase (DLST), the E2k component of human alpha-ketoglutarate dehydrogenase complex (KGDHC), in the AD3 candidate region using yeast artificial chromosomes (YACs). The DLST gene is a candidate for the AD3 gene since deficiencies in KGDHC activity have been observed in brain tissue and fibroblasts of AD patients. The 15 exons and the promoter region of the DLST gene were analysed for mutations in chromosome 14 linked AD cases and in two series of unrelated early-onset AD cases (onset age < 55 years). Sequence variations in intronic sequences (introns 3, 5 and 10) or silent mutations in exonic sequences (exons 8 and 14) were identified. However, no AD related mutations were observed, suggesting that the DLST gene is not the chromosome 14 AD3 gene.

Selection of human chromosome 21-specific DNA probes for genetic analysis in Alzheimer's dementia and Down syndrome

SummaryWe used a mouse-human somatic cell hybrid to construct a chromosome 21-enriched library in phage vector EMBL4. In all, 35 phage clones containing human inserts were identified by differential screening with total human and mouse DNA. Whole recombinant phages were regionally mapped on chromosome 21 by Southern blot analysis using competitive hybridisation conditions to block repetitive sequences. Ten phage clones mapped proximal to a translocation breakpoint in band 21q21.2, while 25 mapped distal to this point. Three of the phage clones identify restriction fragment length polymorphisms. Polymorphic chromosome 21 markers may be useful in the genetic analysis of Alzheimers dementia and Down syndrome.

Determination of the genomic organization of human presenilin 1 by fiber-FISH analysis and restriction mapping of cloned DNA

The presenilin 1 gene (PSEN1) is the most frequently mutated gene in familial early-onset Alzheimer’s disease (AD; Cruts et al. 1998). AD is the most common form of dementia due to neurodegeneration of the central nervous system. The autosomal dominant form of AD is extremely rare, since most AD patients are sporadic cases with no family history of disease. However, genetic association studies using polymorphisms within or near PSEN1 have suggested that PSEN1 might also be a risk factor for nonfamilial AD (reviewed by Cruts and Van Broeckhoven 1998). The exact nature of the genetic cause underlying the association is not known. PSEN1 was localized on Chr 14q24.3 and identified by a positional cloning strategy (Sherrington et al. 1995). PSEN1 is highly homologous to presenilin 2 (PSEN2; 63% sequence similarity in coding exons; Cruts et al. 1996), which is located on Chr 1q42.1 and is also implicated in autosomal dominant early-onset AD (reviewed by Cruts and Van Broeckhoven 1998). The role of PSEN2, however, seems less important in AD, since only few mutations have been identified, and genetic association with AD could not be demonstrated (Cruts et al. 1998; van Duijn et al. 1998). Several transcripts of PSEN1 were identified (Sherrington et al. 1995), the major transcript of which is 2.7 kb. PSEN1 codes for a protein of 467 amino acids. The PSEN1 protein (psen1) is an integral membrane protein with six or eight transmembrane domains (reviewed by Cruts et al. 1996). Psen1 localizes to the endoplasmic reticulum and the golgi complex. Its exact biological function is not yet clear; however, homology searches identified two other integral membrane proteins in Caenorhabditis elegans (C. elegans ), spe-4 and sel-12. Spe-4 is involved in intracellular protein transport during spermatogenesis, while sel-12 facilitates lin-12-mediated Notch cell signaling during development. The PSEN1 gene consists of 13 exons with exon 1A, 1B, and 2 being 58 untranslated region (UTR) exons (Clark et al. 1995; Rogaev et al. 1997). The translation initiation and termination codons are located in exons 3 and 12 respectively. Exons 1A and 1B are probably alternatively transcribed, since cDNA clones from a human hippocampus cDNA library had exon 1A always spliced to exon 2 (Rogaev et al. 1997). However, information concerning tissue-specific distribution of exon 1Aand exon 1B-containing transcripts is not available. PSEN1 is contained within the phage artificial clone (PAC) 54D12, the insert of which measures 190 kb (Rogaev et al. 1997), but its exact genomic size has not been determined. Also, a partial restriction map of PSEN1 and its promoter region was provided, but this map lacks information about intron sizes or genomic organization of the exons (Rogaev et al. 1997). Previously, we contributed to the positional cloning of PSEN1 by genetic linkage studies and physical mapping of the candidate region, using yeast artificial chromosome (YAC) clones (Cruts et al. 1995a, 1995b). A minimal YAC clone contig of YACs 788H12, 964E2 and 797D11, was identified (Cruts et al. 1995b). When PSEN1 was cloned by Sherrington and associates (1995), we showed that PSEN1 was contained in YAC clone 788H12 (Cruts et al. 1995b). In this study, a cosmid library of YAC 788H12 was constructed by subcloning total yeast DNA in sCOGH2 (Datson et al. 1996). Screening of 2000 clones with the PSEN1 cDNA resulted in 14 PSEN1-positive cosmids, 7 of which were confirmed by hybridization and PCR amplification of PSEN1 exons. Two cosmids that were positive only with PCR amplification and not with Southern blot hybridization were excluded from further experiments. The remaining cosmids contained PSEN1 exons 4–12 with cosmid I17 (exons 4 and 5), S13 (exons 6–9), S40 and F12 (exons 9–12; Fig. 1). The Chr 14q24.3 localization of the cosmids was confirmed by metaphase fluorescent in situ hybridization (FISH) (data not shown). There was no overlap in exon content between I17 and S13. For these cosmids, overlap of intronic sequences could be demonstrated by fiber-FISH and was estimated to be less than 1 kb (Fig. 2a). Overlap between the other cosmids was estimated by fiber-FISH at 8 kb for S13 and S40, and 13 kb for S13 and F12 (data not shown). A fiber-FISH analysis of the 3 cosmids I17, S13, and F12 is illustrated in Fig. 2a. Fiber-FISH of I17 indicated that this cosmid contained less human DNA than predicted from its insert length of 30 kb, which we showed to contain 18 kb ofEscherichia coli(E. coli) DNA (data not shown). Screening of the YAC 788H12 cosmid library with PCR amplification products of PSEN1 exons 1A, 1B, and 2 was negative. Therefore, PAC 54D12 was subcloned in the plasmid vector pGEM7-Zf(+), and the sublibrary was screened with the 5 8 UTR exon probes. Two plasmids were selected containing respectively exons 1A and 1B (B22), and exons 2 and 3 (G21). In addition, we used YERK, a plasmid containing exons 1A–3. This plasmid was obtained by end rescue cloning of YAC Fex3 obtained by fragmenting YAC 788H12 with PSEN1 exon 3 as a target sequence (Del-Favero et al., in press). Since none of the plasmid clones contained exon 4, possibly a gap remained between the plasmid contig (exons 1A–3) and the cosmid contig (exons 4–12). FiberFISH of YERK, I17, and S13 confirmed that the plasmid and cosmid contig did not overlap (Fig. 2b). To close the gap, PAC 54D12 was subcloned in the MluI site of pBACe3.5, and the BAC library was screened with PSEN1 exons 3 and 4. BAC16 was selected and shown by PCR to contain exons 1A–5 (Fig. 1). Cohybridization of B22 and G21 with BAC16 on human genomic DNA fibers showed that the 5 8UTR of the PSEN1 gene is located approximately in the middle of BAC16 (data not shown). Hybridization of DNA fibers with BAC16, plasmid B22, and the two cosmids S13 and S40 confirmed the continuity of the PSEN1 Correspondence to: C. Van Broeckhoven Mammalian Genome 10, 410–414 (1999).

Unique sequence homology in the pericentromeric regions of the long arms of chromosomes 13 and 21

We previously isolated two polymorphic chromosome 21q probes, pVC1.21c (D21S190) and pVC1.34a (D21S149), localized in 21qcen-21q21.2. In addition, pVC1.21c recognized a sequence in 21q22.1-q22.2 and both probes cross-hybridized with non-chromosome-21 sequences. In this study we refined the proximal 21q locations of probes pVC1.21c and pVC1.34a to 21q11.1 and demonstrated that they recognize sequences on chromosome 13 but not on chromosomes 14, 15, and 22. Furthermore, the polymorphisms associated with the two loci were assigned to pericentromeric 13q for pVC1.34a and distal 21q for pVC1.21c. Our results are indicative of a region of unique sequence homology in the pericentromeric region of the long arms of chromosomes 13 and 21.

Genetic Linkage Analysis in Two Large Belgian Alzheimer Families with Chromosome 21 DNA Markers

We previously reported two large Belgian Alzheimer families, AD/A and AD/B, with autosomal dominant transmission of the disease and early onset of the disease symptoms (< 35 years). Both pedigrees were used to examine the distance between the FAD and APP gene, using informative haplotypes of two DNA polymorphisms of the APP cDNA. Furthermore, we analysed possible linkage of both families with chromosome 21 using the markers D21S16 and D21S1/S11 and several other 21 probes. We found positive LOD scores with D21S13. Pulse field gel electrophoresis helped us to prove that D21S13 and D21S16 are closely related and may in fact be regarded as one locus in the linkage analysis of Alzheimer families. Linkage data indicate that the FAD gene is most likely located near D21S13/S16, closer to the centromere.

Identification of chromosome 21 DNA polymorphisms for genetic studies in Alzheimer's disease and Down syndrome

SummaryLinkage studies in families with presenile onset of Alzheimers disease (AD) indicated the presence of a predisposing gene on the proximal long arm of chromosome 21. We mapped four new loci in the candidate AD region using somatic cell hybrids. For three of the four loci, several restriction fragment length polymorphisms were found; for one locus, a multiallelic (CA)n dinucleotide polymorphism was detected. Preliminary genetic mapping of the new polymorphic loci relative to the AD-linked loci was obtained in a reference pedigree. In addition, we used the (CA)n dinucleotide polymorphism to reconstruct the non-disjunction event in a Down syndrome (DS) patient whose mother died of familial AD.

Absence of genetic linkage of Charcot‐Marie‐Tooth disease (HMSN Ia) with chromosome 1 gene markers

We previously reported a large Charcot-Marie-Tooth family not linked to the Duffy blood group marker, supporting the existence of genetic heterogeneity in this neuropathy. In order to investigate the possibility of another disease locus on chromosome 1, we analyzed this family further, using DNA polymorphisms of 6 genes. Absence of linkage makes a second disease locus on chromosome 1 unlikely.

The pericentromeric 21 DNA marker pGSM21 (D21S13) contains an expressed HTF island

The DNA marker locus D21S13, localized in the 21q11.1-q21 region, has been closely linked to familial Alzheimers disease. We constructed a physical map of 1.7 Mb around D21S13 using probes pGSM21 and pGSE9. The results indicated that pGSM21 contains recognition sites for at least three rare-cutting restriction enzymes. The clustering of rare-cutting restriction sites is indicative of the presence of an HTF (HpaII tiny fragment) island. Restriction site mapping and methylation analysis proved that pGSM21 contains a methylation-free HTF island. Furthermore, a cDNA correlate has been isolated confirming that pGSM21 is part of an expressed sequence. Today, the gene associated with pGSM21 is the gene closest to the centromere on the 21q arm.