Twenty-two patients were referred for mutation screening in the genes APP
during the period of April 2008 to June 2010 at the Dept. of Geriatric Medicine, Karolinska University Hospital, Sweden. The screening involved genomic sequencing of exons 16-17 in APP
, exons 2-12 in PSEN1
, exons 3-7 and exon 12 in PSEN2
. No variation in APP
was detected in any of the 22 DNA samples. In contrast we identified polymorphic SNPs in both PSEN1
. Polymorphic SNPs in PSEN2
were frequently detected and, in addition to the seven previously reported polymorphisms, we also found a novel variation at IVS6 +91 (G>C; GRCh37:1:227075950) (see Additional file 3
and Additional file 4
). The functional implication on pathogenesis was tested by in silico
methods. We were not able to detect a functional impact of the variation, neither by splice-prediction analysis nor by examining conservation between species (Additional file 4
). Taken together, our sequencing effort did not find variations that could explain the diseased state of the patients.
To address the possibility of a duplication of APP, two microsatellite markers GDB:196999, (APP-dint), and GDB:188463, (D21S265) were PCR amplified with fluorescent primers. When analyzing the 22 DNA samples, 16 were heterozygous (polymorphic) for the marker GDB:196999 and 20 were heterozygous for marker GDB:188463. In our set-up, the size of alleles for marker GDB:196999 ranged from 163 bp to 191 bp, and the size of alleles for GDB:188463 ranged from 241 bp to 255 bp (Table ). The allele-ratio calculated for marker GDB:196999 was between 0.8 - 1.4 indicating a balanced diploid copy-number, except for sample D08 (Table ). In sample D08, the areas for alleles 174 bp and 189 bp for GDB:196999 had a ratio of 2.24 (17752/7932) indicating an unbalanced 2:1 copy-number (Figure ). Six of the individuals were monoallelic for the marker. When calculating the allele-ratios for marker GDB:188463 in the sample-set, thirteen out of 22 DNA samples had ratios between 0.8 - 1.4. Seven samples had ratios between 1.4 - 1.8 and five of these samples had alleles that only differed by two nucleotides in size (D03, D18, D20 - 22); thereby leading to inflation of the area of the shorter allele (Table ). The ratios for D13 and D15 were slightly higher than 1.4 and monoallelic for GDB:196999. Two samples were monoallelic for marker GDB:188463 (D08 and D16) and therefore did not generate any allele ratios. As an example, in Figure , DNA sample D07 carries two alleles for both markers, GDB:196999 and GDB:188463, and the calculated ratio for alleles GDB:196999-174 and GDB:196999-189 was 1.24, (3213/2582), indicating a balanced copy-number of two. Similarly, the ratio for the alleles of GDB:188463 is calculated to be 1.02 (902/883) for sample D07. Also illustrated, sample D08 was not informative for marker GDB:188463 since it only carried one allele, the 247 bp (Figure ), but an unbalanced copy-number are visible as the ratio, 2.24 (17752/7932), for the alleles of marker GDB:196999. Furthermore, sample D09 was not informative for marker GDB:196999 since it only carried one allele (189 bp), but could be examined at the GDB:188463 marker, with a ratio of 1.08 (6683/6190) (Figure ). The control DNA carrying trisomy 21 displayed three alleles for both examined markers (Figure ). The finding indicated that sample D08 carries a duplication of the genomic locus for marker GDB:196999.
Copy-number determination in 22 AD patients referred for mutation screening.
Figure 1 Electropherograms of microsatellite markers for three different DNA, (D07-D09), and for trisomy 21-DNA control (T21). On the y-axis is peak height and on the x-axis is the marker size in base-pairs. Marker alleles show up as major peaks and indicated (more ...)
To confirm the APP copy-number detected by microsatellite analysis, we used a quantitative real-time PCR method (The TaqMan Copy Number Assay, Applied Biosystems Carlsbad, California). The number of copies was determined by comparing patient DNA with a positive control DNA from a subject with trisomy 21 (copy-number of three). Three different exons of the APP gene were amplified by a duplex real-time PCR method for exon 1, exon 7, and exon 17. By calculating the relative copy-number, samples D07 and D09 were found to be diploid for all three amplicons (Figure , top). In contrast, sample D08 was found to have a copy-number value of three for all three amplicons. Thus, the copy-number assay confirmed the presence of three copies detected by the microsatellite analysis for D08. All the other DNA samples were determined by the copy-number assay to have relative copy-number values of 1.52-2.33, indicating a copy-number of two (Table ). Noteworthy, the copy-number assay for samples D13 and D15 showed no variation in any of the exons examined and thus could be determined to carry normal number of the APP gene.
Figure 2 Bar graph showing copy-number from the copy-number assay targeting APP exons (top) and genes flanking APP gene (lower). On the y-axis is the copy-number calculated relative to trisomy 21-DNA control, and on the x-axis are three patient DNA samples, D07, (more ...)
We wanted to address how far the duplication extended into the flanking chromosomal regions and if any neighboring genes were duplicated in addition to APP
. For this purpose, copy-number assays targeting the genes MIR155HG
, and ADAMTS1
were analyzed (for locations see Additional file 2
). The relative copy-number for samples D07 and D09 was two for all of the four investigated genes (Figure lower). The relative copy-number values ranged from 1.78 (ATP5J
, sample D07) to 2.15 (ATP5J
, sample D09). The duplication established in sample D08 was determined to include the locus for the ATP5J
gene located centromeric as well as the locus for the CYYR1
gene located telomeric of APP
and the relative copy-number for these two assays were close to three, (3.34 and 3.41 respectively) (Figure lower). However, the duplication did not include the more distal gene MIR155HG
, located centromeric, or the gene for ADAMTS1
located telomeric (Figure lower) as indicated by the relative copy-number values of 2.14 and 2.07 respectively. We could therefore limit the duplication to a maximal region of 1.27 Mb on chromosome 21 between genomic position 25 868029 bp and 27 138783 bp (Build 36.1).
To fine-map the duplicated region, array-CGH was used by hybridizing DNA sample D08 to a 180 k array covering the APP locus. The method compares hybridization signals from the D08 DNA with a diploid control DNA. The signal intensities were plotted against positions on the chromosome and shows that the duplication covers a region on chromosome 21 restricted to a maximum size of 1.09 Mb within coordinates 25 950727 bp - 27 043885 bp, and to a minimum size of 1.01 Mb within coordinates 25 957532 bp - 26 971 366 bp (Figure ). Thus, the array-CGH confirms the presence of a genomic duplication including the APP gene in sample D08.
Figure 3 Plot from array-CGH made with DNA analytics software. On the y-axis is the log2 ratio signal intensity between Cy3-labeled sample D08 and Cy5-labeled diploid control DNA. The x-axis shows the genomic position of annotated genes (Build 36.1). The colored (more ...)