Abstract:
The present invention relates to mutations in the amyloid precursor protein (APP) promoter region, whereby the mutations cause a significant increase in APP expression. The increase in APP expression is related to Alzheimer&#39;s disease, and the mutations can be used in Alzheimer&#39;s disease diagnosis, or in the construction of transgenic animal models for studying Alzheimer&#39;s disease.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This is a national phase entry under 35 U.S.C. §371 of International Patent Application PCT/EP2007/052296 filed Mar. 12, 2007, published in English as International Patent Publication WO 2007/104739 A1 on Sep. 20, 2007, which claims the benefit under Article 8 of the PCT of European Patent Application Serial No. 06111272.8 filed Mar. 16, 2006. 
     TECHNICAL FIELD 
     The present invention relates to mutations in the amyloid precursor protein (APP) promoter region, wherein the mutations cause a significant increase in APP expression. The increase in APP expression is related to Alzheimer&#39;s disease, and the mutations can be used in Alzheimer&#39;s disease diagnosis, or in the construction of transgenic animal models for studying Alzheimer&#39;s disease. 
     BACKGROUND 
     The currently most favored hypothesis advocates a pivotal role for the amyloid precursor protein in the molecular etiology of Alzheimer&#39;s disease. Clinical mutations in autosomal dominant AD were shown to increase relative concentrations of the 42 amino acids amyloid β (Aβ) peptide (Suzuki et al., 1994; Scheuner et al., 1996; Citron et al., 1997), a proteolysis product of APP, that shows increased propensity to aggregate (Burddick et al., 1992) and deposit in amyloid plaques in AD brains (Iwatsubo et al., 1994). Also, different missense mutations in the APP gene (APP) cause autosomal dominant early-onset familial AD (Goate et al., 1991) (Alzheimer Disease &amp; Frontotemporal Dementia Mutation Database). Standard molecular diagnostic screening of APP is currently limited to exons 16 and 17—coding in part for the Aβ peptide—and their flanking splice sites. However, it is yet not excluded that genetic variation influencing transcriptional activity of APP might also contribute to disease risk. Aβ peptide production depends largely on the amount of APP substrate and, therefore, it is conceivable that regulation of APP transcription might indeed play an important role in AD susceptibility. In fact, several studies have identified higher levels of APP mRNA in AD brains (for a review, see Theuns and Van Broeckhoven, 2000), and increased expression of APP has been correlated with Aβ deposition in brain in instances such as severe head injury (Gentleman et al., 1993). Perhaps the most convincing evidence came from the observation that APP triplication in Down syndrome (DS) patients leads to an overexpression of APP (Rumble et al., 1989) and deposition of Aβ peptide in neuritic amyloid plaques (Wisniewski et al., 1985), resulting in a 50 year earlier onset of AD symptoms. 
     APP is expressed in a variety of tissues, with the highest expression levels in neuronal cells of the central nervous system (CNS), and can be induced by a variety of agents such as growth hormones and cytokines as well as stress conditions. Up-regulation of APP transcriptional activity (Siman et al., 1989; Sola et al., 1993) corroborated with the mRNA expression studies (Wirak et al., 1991; Lahiri and Nall, 1995), suggesting a major role for the APP promoter activity in APP expression. The proximal promoter region of APP is devoid of a functional TATA box, shows a high GC content and transcription initiation is regulated by a strong initiator element (Inr) surrounding the major transcription start site (TSS)+1 (Salbaum et al., 1988; La Fauci et al., 1989; Yoshikai et al., 1990; Quitschke et al., 1996). Further, APP promoter activation is mainly governed by two GC-rich elements, the −93/−82 fragment (APBβ) and the −65/−41 fragment (APBα) (Pollwein et al., 1992). Transcriptional activation of APP can also be mediated by heat-shock factor-1 (HSF-1) binding to the heat shock element (HSE) at position −317 following induction by numerous stress factors (Dewji and Do, 1996). Another transcriptional activator was mapped to −350/−366 harboring an AP-1 binding site and flanking the GC box (Querfurth et al., 1999). Of further interest is that members of the NFκβ/Rel family can specifically recognize two identical sequences at −2250/−2241 and −1837/−1822, in the distal promoter region of APP, referred to as APPκβ sites (Grilli et al., 1995). Both the expression patterns and the proximal promoter region of APP are highly conserved between mammalian species (≧80%) (Yamada et al., 1989; Izumi et al., 1992; Chernak, 1993; Song and Lahiri, 1998). 
     Linkage and association studies support that genetic variability at the APP locus might contribute to increased risk for late-onset AD (Pericak-Vance et al., 1991; Kehoe et al., 1999; Wavrant-De Vrieze et al., 1999; Olson et al., 2002; Meyers et al., 2002; Blaker et al., 2003), in the absence of coding mutations (Lidell et al., 1995). These genetic data further suggested that increased susceptibility might result from genetic mutation in the 5′ regulatory region of APP. However, early on screenings of the APP promoter in sporadic and familial early- and late-onset AD patients, did not reveal AD-specific mutations (Lidell et al., 1995; Fidani et al., 1992; Rooke et al., 1992; Rogaev et al., 1993). More recent studies detected a mutation of G to C at position +37 (hereinafter +37G&gt;C) polymorphism in APP exon 1 while sequencing the −573/+125 fragment of the APP promoter in 20 individuals (Athan et al., 2002). The +37 C-allele was overrepresented in patients with late-onset AD lacking apolipoprotein E (APOE) ε4 alleles (17.2%) compared to elderly control individuals (10%) (OR 2.08, CI 1.26-3.45, adjusted for age, gender and education). Subsequent sequencing of the −308/+124 fragment in 173 patients with late-onset AD and 840 control individuals revealed one more rare variant in control individuals, −9G&gt;C (0.7%), but absent in AD patients. However, both variants, −9G&gt;C and +37G&gt;C, showed no allelic differences in promoter activity when tested in U-87 glioma cells using a reporter gene assay. 
     The APP locus is known to be complex with several other active sites in the 5′ regulatory region apart from the core promoter. In fact, functional elements that control activity of the human APP promoter are located in three distal regions −2257/−2234, −2250/−2241 and −1837/−1822. Constructions in which the distal region −2435/−2165 has been removed showed an increase in promoter activity (Lahiri et al., 1999). However, this large deletion is not occurring in Alzheimer patients, and no Alzheimer disease-related mutations could be found in this region. 
     DISCLOSURE OF THE INVENTION 
     Surprisingly, we found several point mutations in the proximal promoter region that cause a significant increase in expression of APP. These mutations occur only in Alzheimer&#39;s disease patients and are clearly associated with the disease. 
     A first aspect of the invention is a mutation in the regulatory region of the amyloid precursor protein gene resulting in at least a 1.2 times increase in amyloid precursor protein expression, preferably a 1.5 times increase in APP expression as compared with wild-type expression. For the use in this application, increase in amyloid expression is measured as luciferase activity, after transfection of APP-promoter luciferase reporter constructs in human SH-SY5Y neuroblastoma cells, as described in the materials and methods to the examples. The regulatory region of the APP gene, as used herein, consists of the region −5529 till +204 of SEQ ID NO:1 (Lahiri, 2004). Preferably, the regulatory region is limited to the region −2257 till +204. Most preferably, the mutation is situated in the APP proximal promoter region (−766/+204). A mutation as used herein is limited to mutations that can be found in patients. Preferably, the mutation is a small deletion (less than ten nucleotides), an insertion such as a partial duplication within the regulatory region, or a point mutation. Even more preferably, the mutation is a point mutation. More preferably, the mutation is selected from the group consisting of −118C&gt;A, −369C&gt;G, −479C&gt;T and −534G&gt;A. Most preferably, the mutation is selected from the group consisting of −118C&gt;A, −369C&gt;G and −534G&gt;A. 
     Another aspect of the invention is the use of a mutation according to the invention in diagnosis of Alzheimer&#39;s disease. Any method capable of detecting the mutation can be used. Methods to identify the mutation in patient material are known to the person skilled in the art and include, but are not limited to, PCR amplification, sequencing and hybridization. 
     Still another aspect of the invention is the use of a mutation according to the invention in the construction of a cell line. The cell line will be overproducing APP and can be used to study the amyloid formation and/or to test compounds preventing amyloid formation. 
     Still another aspect of the invention is the use of a mutation according to the invention in the construction of a non-human transgenic animal. Indeed, introduction of a mutation according to the invention in a model organism such as a mouse would lead to an increase of APP production and development of an Alzheimer&#39;s disease-like phenotype. Methods to make mutant APP-based transgenic animals have been described, amongst others, in GB2380196 and in WO9640895. Such model organisms could be used for screening compounds and testing medication useful for treatment of Alzheimer&#39;s disease. 
    
    
     
       DESCRIPTION OF THE DRAWINGS 
         FIG. 1 : Conservation plot of the APP 5′ upstream region. Plots were generated using the VISTA Browser software and tracks were presented on the UCSC browser (Frazer et al., 2004). Conserved regions are defined as regions with a conservation score 50% or higher that are larger than or equal to 20 bp. Horizontal black lines crossing the conservation plots mark the 70% conservation boundary. Regions of high conservation are colored according to the annotation as exons (dark blue), UTRs (light blue) or non-coding sequences (pink). 
         FIG. 2 : Transcriptional activity of APP promoter variants. Panel A: Bars represent firefly/ renilla  luciferase ratios for the different constructs (relative luciferase activity, RLA). Values are mean±SEM of at least eight independent measurements. The significance of differences in expression was calculated using the Mann-Whitney U test and p-values are presented above the bars. Panel B: Relative increase of APP promoter activity compared to WT. 
         FIG. 3 : Allele-specific binding of transcription factors. EMSA analysis of allele-specific effect of A) −118C/A, B) −369C/G, and C) −534G/A on the interaction of nuclear protein complexes extracted from SH-SY5Y cells. DIG-labeled double-stranded probes (200 fmol) were incubated with 10 μg nuclear extract from SH-SY5Y cells. In competition experiments, a 50-fold excess of unlabeled probe was added prior to the addition of the labeled probes. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     EXAMPLES 
     Materials and Methods to the Examples 
     Patient-Control Groups 
     AD patients (N=180) and control individuals from the Dutch-speaking Flanders region in Belgium were derived from a prospective study of dementia (Engelborghs et al., 2003; Engelborghs et al., 2006), whereas Dutch patients (N=111) and control individuals were ascertained in a population-based study of early-onset AD in the four northern provinces of The Netherlands and the area of metropolitan Rotterdam. The patients were sampled during two study periods. The original sample was collected between 1980 and 1987 (Hofman et al., 1989) and was extended between 1997 and 2000 in a genetically isolated part of the previously described area with the same sampling criteria (Dermaut et al., 2003). Main characteristics of these study samples are summarized in Table 1. Dutch patients were diagnosed with probable AD before 65 years. Belgian AD patients were included till an onset age of 70 years; 80 patients had an onset age of ≦65 years. Clinical diagnosis of probable AD was based on consensus by at least two neurologists in the Belgian study, or a neurologist and a member of the research team in the Dutch study, according to the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer&#39;s Disease and Related Disorders Association criteria (McKhann et al., 1984). For all patients, detailed data on family history of dementia in first-, second-, and third-degree relatives were collected by interviewing a next of kin of the patient. The criterion we used for familial patients was at least one first-degree relative with dementia. The criteria for autosomal dominant inheritance were (1) at least three patients with clinically diagnosed AD in two or more generations and (2) detailed medical records available on the clinical diagnosis of AD in at least two affected relatives. 
     
       
         
               
             
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Characteristics of Belgian and Dutch early-onset AD study groups 
               
             
          
           
               
                   
                 Belgian sample 
                 Dutch sample 
               
             
          
           
               
                   
                   
                 Control 
                   
                 Control 
               
               
                   
                 AD 
                 subjects 
                 AD 
                 subjects 
               
               
                   
               
             
          
           
               
                 Number 
                 180 
                 180 
                 111  
                 270 
               
               
                 Mean AAO/AAI ± SD 
                 63.8 ± 5.9 
                 59.2 ± 16.7 
                 56.1 ± 5.5 
                 57.5 ± 2.8 
               
               
                 (years) 
               
               
                 Female (%) 
                 55 
                  56 
                 76 
                  60 
               
               
                 Familial AD (%) 
                 41.5 
                 n.a. 
                 69 
                 n.a. 
               
               
                   
               
               
                 Note: 
               
               
                 AAO, age at onset; 
               
               
                 AAI, age at inclusion; 
               
               
                 SD, standard deviation; 
               
               
                 n.a., not applicable; 
               
               
                 “familial” was defined as having at least one affected first degree relative. 
               
             
          
         
       
     
     Belgian (N=180) and Dutch (N=270) control individuals had no neurological or psychiatric antecedents and consisted of subjects without organic disease involving the central nervous system based on clinical examination. Genomic DNA of patients was systematically screened for mutations in the coding exons of four dementia genes PSEN1 [MIM 104311], PSEN2 [MIM 600759], MAPT [MIM 157140] and PRNP [MIM 176640] and exons 16 and 17 of APP [MIM 104760]. We identified putative causal missense mutations in eight Belgian patients (4%, i.e., four in PSEN1, three in PSEN2 and one in APP (Brouwers et al., unpublished data)), and in seven Dutch patients (6%, five in PSEN1 and two in PSEN2) (Dermaut et al., 2003; Cruts et al., 1998). 
     Sequence Analysis of the APP 5′ Regulatory Region 
     The proximal promoter region of APP (−766/+204) was amplified by PCR using three overlapping primer sets: APP-766F (5′-cccccgccccgcaaaatc-3′ (SEQ ID NO:2)) and APP-218R (5′-tgggcttcgtgaacagtgggagggagag-3′ (SEQ ID NO:3)) APP-356F (5′-atgattcaagctcacggggacgag-3′ (SEQ ID NO:4)) and APP-25R (5′-gctcagagccaggcgagtcagc-3′ (SEQ ID NO:5)), APP-99F (5′-ggcggcgccgctaggggtctct-′3 (SEQ ID NO:6)) and APP+204R (5′-ctccagcgcccgagccgtccag-3′ (SEQ ID NO:7)). The distal promoter fragments (−2634/−2159 and −2096/−1563) were amplified using two additional primer sets: APP-2634F (5′-gacgcaatcagcagcataatca-3′ (SEQ ID NO:8)) and APP-2159R (5′-ctgggaaggaggaggcaact-3′ (SEQ ID NO:9)), APP-2096F (5′-catgcttggtttaacgctctgc-3′ (SEQ ID NO:10)) and APP-1563F (5′-gttcactttctgcaccacatttacc-3′ (SEQ ID NO:11)). Oligonucleotide primers for PCR amplification of the APP promoter were based on Genbank D87675.1. Numbering is relative to the major TSS +1 at nt 9001 in D87675.1. About 20 ng genomic DNA were amplified in a total reaction volume of 25 μl containing 10 pmol of each primer, 0.2 mM dNTPs (dATP, dCTP, dTTP) (Amersham, Buckinghamshire, UK), 0.5 mM 7-Deaza-dGTP (Amersham), 0.5 U Platinum or Titanium Taq DNA polymerase (Invitrogen, Carlsbad, Calif. USA) and 1× Platinum/Titanium Taq reaction buffer. Further reaction conditions were thoroughly optimized for each primer set (available upon request). PCR products were screened for mutations by direct sequencing using BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, Calif.) on the ABI3730 automated DNA sequencer (Applied Biosystems) using the PCR primers. Sequences were analyzed using the NovoSNP software (Weckx et al., 2005). Each of the variations was confirmed by RFLP or pyrosequencing (Table 2) in the respective carriers. To screen 450 age- and gender-matched healthy controls, we used deaza-sequencing to detect the variations located between −766 and −218 and pyrosequencing assays for the remaining variations (Table 2). Due to the high GC content of the APP promoter, it was necessary to perform a nested PCR with the pyrosequencing primers on the respective promoter PCR products. 
     MatInspector (Quandt et al., 1995) was applied to investigate the effect of the variations on putative transcription factor binding sites using a core similarity cut-off value of 0.75 and an optimized matrix similarity threshold. Conserved sequences were detected using the VISTA tools. 
                                   TABLE 2                   Mutation detection assays.            Variation   PCR primers   Pyrosequencing/Sequencing primers               −2335 C&gt;T   5′-gacgcaatcagcagcataatca-3′   5′-bio-gatctcggctcacttcaagc-3′           (SEQ ID NO: 12)   (SEQ ID NO: 13)           5′-ctgggaaggaggaggcaact-3′   5′-aaattagccgggcgtcgt-3′           (SEQ ID NO: 14)   (SEQ ID NO: 15)               5′-gtagtcccagctac-3′               (SEQ ID NO: 16)               −1901G&gt;T   5′-catgcttggtttaacgctctgc-3′   5′-attctcctgcctcagcctct-3′           (SEQ ID NO: 17)   (SEQ ID NO: 18)           5′-gttcactttctgcaccacatttacc-3′   5′-bio-gtgaaacccccatctctactaaaaat-3′           (SEQ ID NO: 19)   (SEQ ID NO: 20)               5′-gctgggattacaggca-3′               (SEQ ID NO: 21)               −1750G&gt;A   5′-catgcttggtttaacgctctgc-3′   5′-bio-cctgacctcaggtgatctgc-3′           (SEQ ID NO: 22)   (SEQ ID NO: 23)           5′-gttcactttctgcaccacatttacc-3′   5′-gcaaacgtgagaccctttgt-3′           (SEQ ID NO: 24)   (SEQ ID NO: 25)               5′-attattaagaattttaaggc-3′               (SEQ ID NO: 26)               −534G&gt;A       5′-gaaattccaggttgctcgtg-3′               (SEQ ID NO: 27)               5′-bio-ggcgtttctggaagagaatg-3′               (SEQ ID NO: 28)               5′-gggggttaaaaaatgag-3′               (SEQ ID NO: 29)               −479C&gt;T       5′-ctgtctcaacaagcaaagaaaatcct-3′               (SEQ ID NO: 30)               5′-bio-gtggggcaggcgtttctg-3′               (SEQ ID NO: 31)               5′-ttaagcttcactcgtt-3′               (SEQ ID NO: 32)               −371G&gt;A,   5′-cccccgccccgcaaaatc-3′   5′-cccccgccccgcaaaatc-3′       −369C&gt;G   (SEQ ID NO: 33)   (SEQ ID NO: 34)           5′-tgggcttcgtgaacagtgggagggagag-3′   5′-tgggcttcgtgaacagtgggagggagag-3′           (SEQ ID NO: 35)   (SEQ ID NO: 36)               −118C&gt;A   5′-atgattcaagctcacggggacgag-3′   5′-bio-agggcgctgcacctg-3′           (SEQ ID NO: 37)   (SEQ ID NO: 38)           5′-gctcagagccaggcgagtcagc-3′   5′-ctcggcacccgagaga-3′           (SEQ ID NO: 39)   (SEQ ID NO: 40)               5′-gaactgcgcccgct-3′               (SEQ ID NO: 41)               +37G&gt;C   5′-ggcggcgccgctaggggtctct-3′   5′-bio-gggctccgtcagtttcct-3′           (SEQ ID NO: 42)   (SEQ ID NO: 43)           5′-ctccagcgcccgagccgtccag-3′   5′-ccgcgtccttgctctg-3′           (SEQ ID NO: 44)   (SEQ ID NO: 45)               5′-gggcccccgcgca-3′               (SEQ ID NO: 46)               −371G/A and −369C/G were detected by sequencing and confirmed by StuI RFLP. For all other variations, a Pyrosequencing assay was designed.            
Luciferase Reporter Gene Constructs
 
     Genomic fragments of the APP proximal promoter were obtained by PCR amplification of DNA of patients or asymptomatic mutation carriers using the APP−766F and APP+204R primers as described above and cloned into the pCR2.1-TOPO vector (Invitrogen). The integrity of all inserts was confirmed by sequence analysis using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, Calif.) using vector-specific primers. Mutant clones were selected and consequently recloned into the promoterless pGL3 basic vector (Promega) upstream of the firefly luciferase gene, using KpnI and XhoI. 
     Transient Transfection in Cultured Mammalian Cells 
     Human SH-SY5Y neuroblastoma cells were propagated in a minimal essential medium with Earle&#39;s salt, 10% fetal bovine serum, 2 mM L-glutamine, 200 IU/ml of penicillin, 200 g/ml of streptomycin and 0.1 mM nonessential amino acids (Invitrogen). Human HEK293 embryonic kidney cells were propagated in OptiMem (Invitrogen) with 10% fetal bovine serum, 200 IU/ml of penicillin and 200 g/ml of streptomycin. For transient transfection, SH-SY5Y and HEK293 cells were seeded in 24-well tissue culture dishes, at 7.5×10 5  and 6×10 5  cells/well, respectively, and allowed to recover for 24 hours. Cells were co-transfected with 32 ng (HEK293) or 80 ng (SH-SY5Y) of pRL-TK plasmid containing the herpes simplex virus thymidine kinase promoter upstream of the  renilla  luciferase gene (Promega) and 800 ng of either one of the APP promoter constructs or one of the control plasmids, using 2.4 μl Lipofectamine 2000 (Invitrogen). Empty pGL3-basic vector was used as a negative control, pGL3-promoter plasmid containing the SV40 early promoter upstream of the firefly luciferase gene (Promega) as a positive control. 
     Luciferase Activity 
     Transfected cells were cultured for 24-36 hours, washed with 1 ml phosphate-buffered saline (PBS, Invitrogen), and lysed with Passive lysis buffer (Promega). Firefly luciferase activities (LA F ) and  renilla  luciferase activities (LA R ) were measured sequentially using a Dual-Luciferase reporter assay system (Promega) and a Veritas™ Microplate Luminometer with Dual Reagent Injectors Luminometer (Promega). To correct for transfection efficiency and DNA uptake, the relative luciferase activity (RLA) was calculated as RLA=LA F /LA R . 
     Electrophoretic Mobility Shift Assays 
     Nuclear factors were extracted from SH-SY5Y cells using the NucBuster Protein Extraction Kit (Novagen). DIG-labeled single-stranded oligonucleotides of 31 bp spanning each variant of APP −534G/A, −369C/G and −118C/A were designed and HPLC purified. Blunt-ended double-stranded probes were obtained by annealing of the specific oligonucleotides with their respective reverse complements and checked on a non-denaturing 15% polyacrylamide gel in 0.25×TBE. For the binding reactions, 200 fmol DIG-labeled double-stranded probe was added to a total reaction volume of 20 μl containing 10 μg SH-SY5Y nuclear extract, 1× binding buffer (12% glycerol, 20 mM HEPES or TRIS, 50 mM KCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF) and 1 μg poly (dI-dC) (Roche Applied Science). For competition assays, unlabeled double-stranded probes were added to the reaction mixture prior to addition of the labeled probe. Binding reactions were incubated at room temperature for 20 minutes. Protein-DNA complexes were analyzed by electrophoresis on non-denaturing 6% polyacrylamide gels in 0.25×TBE and visualized by chemiluminescent detection using the DIG gel shift kit (Roche Applied Science). 
     Real-Time PCR mRNA Quantification 
     mRNA was isolated from cultured lymphoblast cells of mutation carriers and control individuals using the mRNA Chemagic isolation system (Chemagen, Baesweiler, Germany) and first strand cDNA was synthesized from 300 ng of mRNA using the SuperScript™ III First-Strand Synthesis System for RT-PCR (Invitrogen). APP expression levels were quantified using a Taqman-MGB real-time PCR assay on the ABI Prism 7900HT Sequence Detection System (Applied Biosystems). Primers and probes were designed with the PrimerExpress software (Applied Biosystems) and sequences are available upon request. Human APP mRNA quantities were normalized for the three housekeeping genes, ubiquitine C (hUBC), β2-microglobulin (hB2M) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (hYWHAZ (SEQ ID NO:47)) as recommended by Vandesompele et al. (2002). 1/20 fold-diluted cDNA was amplified in a 20 μl reaction containing 1×qPCR Mastermix Plus (no UNG) (Eurogentec, Seraing, Belgium), 300 nM primers and 250 nM probe using the universal amplification protocol (Applied Biosystems). 
     Statistical Analysis 
     The Mann-Whitney U test, a nonparametric analogue to the unpaired t test, was used to compare the RLA produced by the wild-type and mutant transfectants, as well as real-time PCR quantifications of APP expression levels. 
     Example 1 
     Variation in the 5′ Regulatory Region of APP 
     We sequenced the −766/+204 region of the proximal APP promoter and exon 1, and two more distal promoter regions −2634/−2159 and −2096/−1563, in two independently ascertained Dutch-speaking early-onset AD groups composed of 180 Belgian and 111 Dutch patients (Table 1). In total, we identified nine heterozygous sequence variants and confirmed their presence in the respective carriers by a second method, i.e., PCR-RFLP analysis or pyrosequencing (Table 3—Quandt et al., 1995; Brouwers et al., 2006). Analysis in 450 age- and gender-matched healthy control individuals (180 Belgian and 270 Dutch individuals) demonstrated that three variants (33%) were known polymorphisms: −2335C/T (rs364091), −1901G/T (rs1235879) and +37G/C (rs459543) (Table 3A), the latter being the same one reported by Athan et al., (2002). Also, in agreement with this study, the −9G&gt;C variant was not observed in the AD patients. Six of nine variations (67%) were present in patients only (Table 3B). One Dutch patient, d807, with onset age 50 years was compound heterozygote for two promoter mutations −1750G&gt;A and −118C&gt;A. 
     
       
         
               
             
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                 Sequence variants in the APP 5′ regulatory region. A. Common 
               
               
                 variants; B. Early-onset AD patients; and C. Control individuals. 
               
               
                   
               
             
             
               
                 A. 
               
             
          
           
               
                   
                   
                   
                 TF binding alterations 2   
               
               
                   
                 Variation 1   
                 Frequency (controls) 
                 (core/matrix similarity) 
               
               
                   
                   
               
               
                   
                 −2335 C/T 
                 4% 
                 No matches 
               
               
                   
                 −1901 G/T 
                 4% 
                 +PAX-6 (0.75/0.75) 
               
               
                   
                   
                   
                 +FKHD (0.75/0.79) 
               
               
                   
                  +37 G/C 
                 4% 
                 +CDEF (1/0.87) 
               
               
                   
                   
               
             
          
           
               
                 B. 
               
             
          
           
               
                   
                   
                   
                   
                 Current 
                   
                   
                   
               
               
                   
                   
                   
                 Onset 
                 or age at 
                   
                   
                 TF binding alterations 2   
               
               
                   
                 Patient 
                   
                 age 
                 *death 
                 Family 
                   
                 (core/matrix 
               
               
                 Variation 1   
                 ID 
                 Nationality 
                 (years) 
                 (years) 
                 history 
                 APOE 4   
                 similarity) 
               
               
                   
               
               
                 −1750G &gt; A 3   
                 d807 
                 Dutch 
                 50 
                 *64 
                 − 
                 34 
                 No major changes 
               
               
                  −534G &gt; A 
                 d768 
                 Dutch 
                 52 
                 *70 
                 + 
                 34 
                 +OCT-1 (0.89/0.92) 
               
               
                  −479C &gt; T 
                 d5165 
                 Belgian 
                 62 
                 *69 
                 − 
                 33 
                 −GAGA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 (0.750/0.789) 
               
               
                   
                   
                   
                   
                   
                   
                   
                 +OCT-1 
               
               
                   
                   
                   
                   
                   
                   
                   
                 (0.771/0.843) 
               
               
                  −371G &gt; A 
                 d4605 
                 Belgian 
                 55 
                  64 
                 − 
                 33 
                 −AP2 (0.976/0.916) 
               
               
                   
                   
                   
                   
                   
                   
                   
                 −STAF (0.904/0.799) 
               
               
                  −369C &gt; G 
                 d811 
                 Dutch 
                 63 
                 *? 
                 + 
                 34 
                 −AP-2 (1/0.92) 
               
               
                   
                 d1081 
                 Belgian 
                 61 
                  75 
                 + 
                 44 
               
               
                  −118C &gt; A 3   
                 d807 
                 Dutch 
                 50 
                 *64 
                 − 
                 34 
                 −AP-2 (1/0.91) 
               
               
                   
                   
                   
                   
                   
                   
                   
                 −HES-1 (0.83/0.92) 
               
               
                   
               
             
          
           
               
                 C. 
               
             
          
           
               
                   
                   
                   
                   
                   
                 TF binding alterations 2   
               
               
                   
                   
                   
                 Age at inclusion 
                   
                 (core/matrix 
               
               
                   
                 Variation 1   
                 ID 
                 (years) 
                 APOE 4   
                 similarity) 
               
               
                   
                   
               
               
                   
                 −375G &gt; C 
                 d3155 
                 56 
                 33 
                 +SP1 (1/0.915) 
               
               
                   
                 −343A &gt; C 
                 d3099 
                 56 
                 33 
                 No alterations 
               
               
                   
                   
               
               
                   
                 Note: 
               
               
                   
                   1 Position relative to the major APP transcription start site (Inr) at nt. 9001 in GenBank accession number D87675.1; 
               
               
                   
                   2 Matinspector analysis (Quandt et al., 1995); 
               
               
                   
                   3 Patient d807 is compound heterozygous for these mutations; 
               
               
                   
                   4 APOE was genotyped as previously described (Brouwers et al., 2006). 
               
             
          
         
       
     
     One other mutation, −369C&gt;G, was identified in two familial patients, d811 (Dutch) and d1081 (Belgian), with very similar onset ages of 63 and 61 years, respectively. Haplotype analysis in these patients identified shared alleles at five neighboring microsatellite markers in the 210 kb APP region suggestive for a potential common ancestor, though the shared haplotype had a frequency of 1% in control individuals. 
     Recently, we obtained autopsied brain material of the Belgian AD patient carrying the APP (−369G&gt;C) promoter mutation and a detailed neuropathological examination is in progress. Structural brain imaging of this patient during the disease progress, however, revealed white matter lesions reminiscent of those found in APP duplication carriers, suffering from AD with pronounced CAA due to overexpression of APP. 
     Another familial Dutch patient, d786, carried the −534G&gt;A mutation. In two Belgian patients, d4605 and d5165, with onset ages 55 and 62 years, the −371G&gt;A and −479C&gt;T mutations were identified, respectively. Neither one of the familial patients fulfilled our criteria for autosomal dominant AD. 
     To investigate to what extent genetic variability in the APP promoter was patient-related, we sequenced the −766/+204 proximal promoter region in 48 control individuals. We identified only two additional rare polymorphisms, −343A&gt;C and −375G&gt;C, that were each present in one control individual (Table 2C). Also, since four of the six mutations identified in patients were located in the −766/−218 region, we sequenced this 548 bp region in all 450 control individuals but did not detect any additional variants. 
     Example 2 
     Transcriptional Activity of APP Promoter Mutations 
     We cloned the −766/+204 APP promoter fragment containing wild-type (WT), or mutant −534A, −479T, −371A, −369G, or −118A alleles into the pGL3basic vector upstream of a firefly luciferase reporter gene. As controls, we used the −375C and −343C alleles, identified in control individuals. Reporter gene assay of transiently transfected human embryonic kidney (HEK293) cells showed no significant differences in expression level between −534A, −479T, −371A, −369G, and −118A and WT promoter sequences. Also, real-time PCR APP mRNA quantification in lymphoblasts from the mutation carriers (N=2) did not reveal significant differences in expression levels compared to control individuals. However, in human neuroblastoma (SH-SY5Y) cells, a significant near two-fold increase (p-value&lt;0.001) in APP transcriptional activity was observed for three of the five mutant alleles identified in early-onset AD patients: −534A, −369G and −118A ( FIG. 2 ). The other two mutant alleles, −479T and −371A, did not significantly increase transcriptional activity (p=0.08 and p=0.8, respectively). Neither one of the two variant alleles found in control individuals significantly altered transcriptional activity of the APP promoter in either one of the studied cell types ( FIG. 2 ). 
     The neuron-specific doubling of expression found caused by the point mutations in the proximal part of the APP promoter is comparable to the increased level of APP expected from the genomic APP triplication in DS patients. Therefore, one can conclude that these promoter mutations also cause a critical elevation of APP in vivo in the AD patients. The reliability of our findings is supported by the fact that all mutation carriers had a follow-up diagnosis of probable AD. Further, in the Belgian prospective study, we observed 100% (N=55), and in the Dutch study, 88% (N=17), correlation with pathological diagnoses in probable AD patients. Also, quantification by ELISA of the abundance of amyloid β, tau and phosphotau in the cerebrospinal fluid (CSF) of two Belgian APP mutation carriers showed a decrease of Aβ42 and a slight increase of tau and phospho-tau typical for AD. 
     Example 3 
     Allele-Specific Transcription Factor Binding 
     We examined whether the three mutant alleles affecting APP expression (−118C/A, −369C/G, −534G/A) interfered with the specific recognition of the APP promoter by nuclear factors extracted from SH-SY5Y cells using electrophoretic mobility shift assays (EMSA). We used 31 bp long double-stranded oligomers containing one of six possible promoter alleles. We observed that SH-SY5Y nuclear extracts contained nuclear proteins binding specifically to all three regions of the APP promoter, resulting in the formation of one or two major complexes ( FIG. 3 ). Competition with 50-fold excess of the respective unlabeled oligomers resulted in complete inhibition of complex formation. For −118C/A, one major complex bound to both alleles, however, with a higher binding affinity for the mutant A-allele. The major complex binding to the −369C/G probe showed a decrease in binding affinity or even a slight shift in complex mobility for the mutant G-allele. Two major complexes were formed on the oligomers of the −534G/A variant, however, with clear differences in binding affinity for the faster migrating complex, which preferentially bound to the WT G-allele. Also, competition with a 50-fold excess of cold −534A oligomer did not completely inhibit binding of the smaller complex to the −534G allele supporting the hypothesis of higher binding affinity of this complex for the G-allele. 
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