Human Molecular Genetics Advance Access originally published online on November 20, 2006
Human Molecular Genetics 2007 16(1):1-14; doi:10.1093/hmg/ddl436
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Genome-wide SNP assay reveals structural genomic variation, extended homozygosity and cell-line induced alterations in normal individuals
1 Molecular Genetics Unit, 2 Unidad de Genética Molecular, Departamento de Genómica y Proteómica, Instituto de Biomedicina de Valencia-CSIC, 46010, Valencia, Spain, 3 Laboratory of Neurogenetics and 4 Retalila Western Institute of Neurological Studies, University College London, London, UK, 5 Department of Neurology, Chang Gung Memorial Hospital and College of Medicine, Chang Gung University, Taipa, Taiwan, 6 Computational Biology Core, National Institute on Aging, 7 Human Motor Control Section and 8 Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA and 9 Coriell Institute for Medical Research, Camden, NJ, USA
* To whom correspondence should be addressed. Tel: +1 3014516079; Fax: +1 3014515466; Email: singleta{at}mail.nih.gov
Received July 31, 2006; Accepted November 11, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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The recent hapmap effort has placed focus on the application of genome-wide SNP analysis to assess the contribution of genetic variability, particularly SNPs, to traits such as disease. Here, we describe the utility of genome-wide SNP analysis in the direct detection of extended homozygosity and structural genomic variation. We use this approach to assess the frequency of genomic alterations resulting from the lymphoblast immortalization and culture processes commonly used in cell repositories. We have assayed 408 804 SNPs in 276 DNA samples extracted from Epstein-Barr virus immortalized cell lines, which were derived from lymphocytes of elderly neurologically normal subjects. These data reveal extended homozygosity (contiguous tracts >5 Mb) in 9.5% (26/272) and 340 structural genomic alterations in 182 (66.9%) DNA samples assessed, 66% of which did not overlap with previously described structural variations. Examination of DNA extracted directly from the blood of 30 of these subjects confirmed all examined instances of extended homozygosity (6/6), 75% of structural genomic alteration <5 Mb in size (12/16) and 13% (1/8) of structural genomic alteration >5 Mb in size. These data suggest that structural genomic variation is a common phenomenon in the general population. While a proportion of this variability may be caused or its relative abundance altered by the immortalization and clonal process this will have only a minor effect on genotype and allele frequencies in a large cohort. It is likely that this powerful methodology will augment existing techniques in the identification of chromosomal abnormalities.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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With the cost of high throughput whole genome SNP genotyping now <11 genotype, public application of genome-wide SNP analysis has begun in earnest. The various methods employed for this approach assay >100 000 SNPs across the genome, selected using both random and targeted (gene-centric, putative functional or haplotype tagging) approaches. This technology has been aimed primarily at cohort-to-cohort analysis to assess the contribution of genetic polymorphism to traits and to disclose details of human evolutionary history, however, it is clear that some of the metrics produced will quickly reveal extended homozygosity and variability in the underlying genomic architecture in individual samples. Previous experiments examining these features have been performed by mining of existing HapMap data (1–3), fosmid end sequencing (4), genotyping with microsatellite markers (5,6) or use of custom manufactured oligonucleotide or construct arrays (7–9). While these approaches have been informative, they do not represent standardized technology which is easily transferable between laboratories.
We embarked on a whole genome SNP genotyping project, focusing on a publicly available cohort of neurologically normal Caucasian individuals aged 55–88 years, and collected from several sites across North America (10). These samples are available from the NINDS-funded Human Genetics Resource Center: DNA and Cell Line Repository at the Coriell Institute for Medical Research (http://ccr.coriell.org/ninds). DNA was extracted from two sources for the genotyping described: from early-passage Epstein-Barr virus immortalized lymphocytes [lymphoblast cell lines (LCLs)] and directly from the blood sample used for each immortalization. In performing these experiments we have provided open data consisting of dense SNP genotypes in a cohort of well-phenotyped, publicly available samples (10).
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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We used the Infinium Human-1 Genotyping BeadChip and the HumanHap300 Genotyping BeadChip (Illumina Inc., San Diego, CA, USA) to genotype DNA extracted from LCLs. These products assay 109 365 gene-centric SNPs (Human-1) and 317 511 SNPs derived from phase I of the International HapMap project (HumanHap300). There are 18 072 SNPs in common between the two arrays; the assays combined provide data on 408 804 unique SNPs.
The raw genotype data from this effort are available on the Coriell website (link available through http://ccr.coriell.org/ninds/catalog). The median genotype success rate was 99.86% (mean 99.73%; range 96.93–99.97%) for samples considered as passing initial quality check. A genotype success rate of less than 95% was considered a failure. Five (1.8%) and eight (2.8%) samples had to be repeated for the Human-1 and HumanHap300 chips, respectively. These samples were repeated using a separately banked DNA aliquot from Coriell Cell Repositories. Three and four of these samples (for Human-1 and HumanHap300 chips, respectively) subsequently gave call rates greater than 95%. Thus, call rates for two of the samples assayed with the Human-1 array (ND01630, ND01666) and four for the HumanHap300 (ND01630, ND01666, ND03447, ND03704) remained below this level: analysis of the results revealed contamination of the DNA stock in these cases. These four samples were dropped from further analysis. Genotyping of six additional replicate samples showed 99.99% call reproducibility among successful genotypes (mean shared success rate of 99.6%). Analysis of the 18 073 SNPs that overlap between the Human-1 and HumanHap300 BeadChip products revealed genotype concordance rates of >99.9% between the assays across 272 samples.
The scan data from each sample was analyzed using the GenomeViewer tool within BeadStudio v2.2.22 (Illumina Inc.). Two metrics were assessed in this utility; log R ratio and B allele frequency. The log R ratio gives an indirect measure of copy number of each SNP by plotting the ratio of observed to expected hybridization intensity. B allele frequency plots the proportion of times an allele is called A or B at each genotype: thus the expected ratios are 1.0 (B/B), 0.5 (A/B) and 0.0 (A/A). These two statistics allow visualization of copy number changes and homozygosity.
Extended homozygosity
We identified 26 samples with contiguous homozygosity tracks greater than 5 Mb long (9.5% of samples assessed; Fig. 1; Table 1). Out of the 26 samples, 11 identified with extended homozygosity (42.3%) showed more than one region of homozygosity greater than 5 Mb in size, 4.5-fold times greater co-occurrence than expected by chance alone. These data are consistent with the view that the observed homozygosity relates to chance meiotic events in subjects with consanguineous parents rather than events related to the LCL creation process or to an artifact of the analytical method.
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Structural genomic alterations
Analysis of log R ratio and B allele frequency in each of the 272 samples assayed with the Human-1 BeadChips revealed data consistent with genomic deletion in nine samples, both sub-chromosomal and affecting the whole chromosome (Table 2). The six observed sub-chromosomal deletions ranged in size from 0.5 to 32.9 Mb. Data from three samples revealed entire genomic deletion of chromosome X. Consistent with simple heterozygous deletion we detected a region 2.6 Mb in size, on chromosome 3 of LCL ND01493, indicated by a decrease in the log R ratio and a lack of heterozygous SNP calls (Fig. 2). For the remaining eight deletions we observed contiguous drops in log R ratio, consistent with a decrease in copy number. However, the observed drop in log R ratio was not coupled with an absence of heterozygous calls in these regions, thus excluding simple, heterozygous genomic deletion (Table 2). Rather, at the sites of interest analysis of B allele frequency revealed four clusters of contiguous genotypes, rather than the expected three. These were at 1 (100% B allele), 0 (100% A allele) and at two other locations, one situated between 0.5 and 1 (cluster c) and one between 0 and 0.5 (cluster d). Within each sample, the average position for cluster c was the same as 1 minus the average position for cluster d, and these cluster positions ranged approximately from B allele frequency 0.15 and 0.85 to 0.45 and 0.55. The observed alterations are consistent with heterosomic deletions, where a heterozygous genomic deletion is present in only a proportion of the cells contained within a sample.
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Analysis of the scan data obtained from each of the 272 samples using the HumanHap300 assay confirmed all these nine deletions and revealed the existence of 119 more, giving a total of 128, of which 28 were heterosomic (Supplementary Material, Table S2).
Results indicative of genomic multiplication were identified in 15 LCL-derived samples after analysis with Human-1 BeadChip: these ranged in size from 0.2 Mb to the entire chromosome, however the majority (13/15) were under 3 Mb in length (Table 2). The profile indicative of a genomic multiplication is an increase in log R ratio in the presence of allele clusters outside the expected homozygous or heterozygous range for the value of B allele frequency (Fig. 3A). As positive controls for the utility of this approach to detect genomic copy number changes, we assayed DNA samples derived from a subject who carries the SNCA locus triplication, 
1.6 Mb in length (11) and a subject with trisomy 21. Data from these samples clearly showed the triplication mutation and the presence of an additional copy of chromosome 21 (Fig. 3B and C). Again, analysis of the scan data of the HumanHap300 BeadChip, confirmed all 15 alterations and revealed 197 more duplications (Supplementary Material, Table S3).
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Comparison of observed deletion and duplication events with those published in the database for genomic variants (http://projects.tcag.ca/variation/) revealed that 34 of 101 identified deletions overlapped with previously reported structural variants and 77 of 212 observed sub-chromosomal multiplications overlapped with previously reported structural mutations.
Comparison of DNA derived from LCLs with DNA extracted from source tissue
In order to confirm that the extended tracts of homozygosity observed were not a result of the creation or passage of LCLs, we repeated the genotyping experiment in six samples using DNA extracted directly from the blood used to generate these lines. This experiment revealed identical patterns of homozygosity between the replicate DNA samples (Fig. 4).
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In an attempt to examine whether the LCL creation and culture process lead to the appearance of the structural genomic variation noted, we re-assayed DNA extracted directly from blood for all samples in which this type of mutation was noted using the Human-1 BeadChip. The blood sample corresponding to LCL ND01493 confirmed the presence of the simple heterozygous deletion in the source tissue. Only one of the eight heterosomic deletions, detected in LCL ND04946, was detected in these replicate experiments (Fig. 5A). Notably the mean cluster positions for clusters c and d described above differed between the DNA extracted from blood and the DNA extracted from the cell line for this subject. These data indicate that this heterosomic deletion was present in the blood of this patient, and that the EBV immortalization process and subsequent culture have altered the ratio of cells with and without the deletion. In this specific instance there is decreased heterosomy in the DNA extracted from the cell line, indicating preferential immortalization and/or culture of cells with a full diploid genome. This region of chromosome 13 covers 32.9 Mb, contains
189 genes, and encompasses the minimal common deleted region described for multiple myeloma and chronic lymphocytic leukemia (12,13). Also of note, two of the heterosomic deletions identified in LCLs but not in blood, were apparently identical in size and position. These were identified in ND01577 and ND03792 at 22q11.2 and span a 0.5 Mb region containing the immunoglobulin lambda gene cluster, a region previously described to be duplicated (8). These events probably represent V(D)J-type recombination either in the LCLs or as a rare event in blood, amplified by the process of LCLs moving toward a clonal state. The five remaining deletions, all apparently heterosomic, were not identified in the original blood samples, suggesting that they were either an artifact of the EBV immortalization process, or a biased representation of the donor tissues state at immortalization, caused by LCL creation and culture.
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Analysis of DNA extracted directly from the blood used for the creation of the LCLs in which genomic duplication was noted revealed 11 of the 15 alterations were also present in the source tissue. The genomic multiplications with discordant results between LCLs and blood comprised four of the five largest multiplications identified (Table 2).
Confirmation of structural alterations by real-time PCR
In order to confirm that the structural alterations found in these samples were not artifacts of the methodological or analytical process, quantitative PCR was performed in all 272 samples using 14 TaqMan probes. This was performed using probes for a 0.5 Mb duplication at chromosome 7q11 (not previously described), a 0.019 Mb deletion in chromosome 11q21 (not previously described), a 33 Mb heterosomic deletion in chromosome 13p12-q21 (overlapping with TCAG's variant number 0313) and a 0.11 Mb duplication in chromosome 16q23.2 (not previously described). In addition, 10 individual probes across the PARK2 locus were used to verify the presence and size of four unique duplications observed in this region in individuals ND01583, ND05536, ND04788 and ND05093. In all instances, structural changes predicted by Infinium analyses were confirmed according to the 2–
Ct value obtained (Figs 6 and 7). The extent of the four unique duplications observed at the PARK2 locus, ranging from 9 to 362 kb in size was confirmed (Fig. 7). No additional carriers of any of these structural changes were found, supporting the sensitivity of this approach.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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We describe here a striking level of homozygosity in a cohort of aged, out-bred North American Caucasians. These data extend upon previous observations of large tracts of homozygosity in subjects typed as part of the HapMap initiative (1) and in subjects from reference families from the Centre d'Etude du Polymorphisme Humain (6). In the analysis of individuals genotyped as a part of the HapMap initiative Gibson and colleagues identified contiguous tracts of homozygosity >5 Mb in length in 3.8% of the subjects assessed (8 of 209) and multiple tracts >5 Mb in half of these subjects (1); our data derived from a single population of aged individuals shows a comparable prevalence of homozygous tracts of 5 Mb and larger (9.5%), and of subjects with multiple homozygous tracts (42.3%). While events such as segmental uniparental disomy could explain our data, the observation that individuals with at least one large tract of homozygosity are likely to harbor other large regions of homozygosity supports the idea that parental consanguinity is the cause. One would presume, given the increased mobility of North Americans in the last 50 years, that analysis of younger subjects would reveal a lower incidence of individuals with extended tracts of homozygosity. These data suggest the utility of whole genome SNP analysis to perform homozygosity mapping of disease loci even in ostensibly out-bred populations. This approach would allow rapid fine-mapping of disease intervals, with the advantage of simultaneously revealing copy number changes often associated with recessive loss of function alleles. In samples from individuals with disease, one would search for the occurrence of overlapping regions of extended homozygosity between different samples from affected individuals.
Previous studies have shown clearly that high density SNP analysis will provide SNPs that act as proxy markers for common structural variation (2,14), however until recently, the use of genome-wide SNP assays as a direct method of determining copy number has not been widely appreciated (15). In the experiments outlined here, we identified genomic copy number changes in 182 samples (340 changes in total). In an effort to demonstrate whether these changes were not a result of the creation or passage of LCLs, we repeated the genotyping experiment in those 24 samples in which alterations were detected with the Human-1 array. Thirteen of the identified changes were readily detected in the source tissue used for immortalization, and concordance rates were higher for smaller structural alterations (75% for those changes <5 Mb; 13% for those changes >5 Mb). Notably, this method discriminates heterosomic and heterozygous genomic deletions, as each allele for each SNP is assayed multiple times. This redundancy can be used to provide a proportion for allele calls at each SNP, thus significant deviation from homozygous (100% allele A or allele B) and heterozygous (50% allele A and 50% allele B) clusters, particularly when observed in multiple contiguous SNPs not only indicates structural alteration but gives a measure of the proportion of cells containing this genomic change. The single simple deletion that we observed was present in both tissues examined. In the case of the identified heterosomic deletions, we can establish these events as somatic, occurring in the source tissue in one instance, with the remaining events resulting from, or emphasized by, the process of LCL creation. An analogous process can be used to assess heterosomic duplications although in this case, the resolution is more difficult because one needs to interpret deviations from a 66%/33% read which is more challenging. Real-time PCR analysis of sample ND04946, which GW-SNP assay suggested included a heterosomic deletion resulting in an 30% drop in copy number, also revealed a drop in copy number of 30%. Previous reports have identified structural alterations in the human genome as small as 1 kb; however, these approaches rely either on the availability of high density parent offspring genotype data (2,3,5), or the use of custom manufactured arrays (7–9). The lower limit in terms of size of structural genomic variability detected in the current study is 0.02 Mb, a limit which is sensitive to the relative density and informativity of SNPs assessed in any one region; thus, the average limit of resolution is likely to be in the range of hundreds of kilobases as is most often seen in the data described here. Although the inability of this method to detect balanced translocations would suggest that this will not yet replace current approaches aimed at defining chromosomal abnormalities, the simple and transferable nature of this technique means it may augment these methods. The cost of initial set-up for applying high-density SNP typing approaches is considerable (hundreds of thousands of dollars), however once established small numbers of samples may be analyzed easily and for relatively little cost, currently ranging from $250 to $700 per sample depending on the platform and SNP density used. Over the next period it is likely that the price will decrease and the SNP density increase. In addition to the usefulness of this technique for quickly mapping structural variability with good resolution, this methodology is appealing because it is easily transferable between laboratories. This would greatly facilitate the rapid accumulation of equivalent data to produce an encyclopedia of normal and abnormal structural genomic variation, similar to previous efforts for gross chromosomal abnormalities (http://jws-edck.wiley.com:8096/) (16) and enable standardized comparison between laboratories.
The utility of this methodology to address other research questions is striking. One can envisage using these assays to catalog the occurrence of both germ-line and somatic structural mutation, both in disease and healthy states. This method also promises to enable genetic characterization of many of the key tools used within laboratories. For example, chromosomal rearrangements have been described in cell lines commonly used in molecular biology laboratories. This may be an evolving process, rather than a stable alteration, and if so, would presumably affect experimental outcome. Routine assay of standard cell lines would allow inter-laboratory standardization. The effect of creating stem cell lines on the genome has not been established; again this technique offers a quick first-pass assay for analyzing the effects of clonally deriving stem-cell lines both for research and for therapies. In all instances the availability of a standardized product to perform these assessments is of great importance. Clearly, a weakness of the current approach is that scoring of structural variability was performed using a largely subjective measure; while we attempted to minimize the false positive and false negative rate by independent calling of structural variation by two experienced data analysts it is likely that some variability was missed. The development of a publicly available tool that uses a statistical basis for the detection of structural variation that can be applied to these data would greatly enhance the ease with which these analyses can be performed.
The present study shows that the relative effects of LCL creation and passage on the genomic architecture of both individual samples and the cohort as a whole are minimal and that the genetic content of LCLs is relatively faithful to that of the starting material used for LCL creation. In the context of a genome wide case–control study the variation which appears to have resulted from, or have been amplified by, the process of creating LCLs is unlikely to significantly interfere with allelic or genotypic associations. This variability is limited to a relatively small number of samples and in these samples the genomic region affected represents a small percentage of the genome. Thus, the aggregate effect of this variability will be to alter <0.1% genotype calls (25 000 individual genotypes potentially affected, from a total of 30 million surveyed). While this bodes well for the use of these data for genetic association studies, further experiments defining the effect of extended culture and passage, if any, will be required to see if this increases structural alteration and potentially effects allele calling.
In conclusion, this study provides public access to whole genome array data in a large series of Caucasians and illustrates that such data is useful, not only for whole genome association studies for which it was planned, but also for determining the extent of inbreeding and structural mutations. Its simplicity suggests unexpected uses for this technology in the assessment of standard laboratory cell lines, stem cells and high resolution cytogenetic testing. Its speed and ease may lead it to replace traditional linkage analysis strategies, especially for recessive diseases but also because it will allow easy assessment of linkage disequilibrium between laboratories without the need for uncertain standardization against CEPH controls. The data presented here also show that the effects of LCL creation and passage on genotypes and genetic architecture are minimal and unlikely to confound the results of a genome-wide case–control study.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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Subject collection
Blood samples for DNA extraction and Epstein-Barr virus immortalization were drawn from unrelated subjects at many different sites within the United States who all underwent a detailed medical history interview. None had a history of neurological illness. All were asked specifically regarding the following disorders: Alzheimer's disease, amyotrophic lateral sclerosis, ataxia, autism, bipolar disorder, brain aneurysm, dementia, dystonia and Parkinson's disease. Folstein Mini-mental state examination scores ranged from 26/30–30/30. All were interviewed for family history in detail. None have any first degree relative with a known primary neurological disorder. Specifically, none have a family history of any first degree relative with any of the following disorders: amyotrophic lateral sclerosis, ataxia, autism, brain aneurysm, dystonia, Parkinson's disease and schizophrenia. For more details see http://ccr.coriell.org/ninds/index.html. Blood was drawn from each subject in two 10 ml acid citrate dextrose tubes following written informed consent to participate.
Sample preparation
Epstein-Barr virus immortalization was performed as previously described (17,18) and DNA was extracted using a modification to the procedure developed by Miller et al. (19) as previously reported. At the same time DNA was extracted from 1/2 ml of blood for subsequent quality control steps in the cell banking process. This series comprises 276 DNA samples (275 unique samples, 1 replicate sample) arrayed on three 96-well plates, NDPT002, NDPT006 and NDPT008. DNA for the primary phase of the experiments was extracted from the Epstein-Barr virus immortalized LCLs. The average passage number for each line was 5 (range 5–7).
Genotyping
All samples within this cohort were genotyped with the Human-1 and HumanHap300 SNP genotyping chips (Illumina Inc.). These products assay 109 365 gene-centric SNPs (Human-1) and 317 511 SNPs derived from phase I of the international HapMap project (HumanHap300). There are 18 072 SNPs in common between the two arrays; the assays combined provide data on 408 804 unique SNPs.
Genotyping was performed as per the manufacturer's protocol (Illumina Inc.) using 750 ng of genomic DNA. Briefly, 750 ng of each DNA sample is isothermally amplified in an overnight step (steps 1 and 2). The amplified product is then enzymatically fragmented, precipitated and resuspended. The resulting product is then hybridized to the chip overnight. The amplified and fragmented DNA samples anneal to locus-specific 50-mers during the hybridization step. Each allele at each locus is represented by one of two bead-types fixed to the chip. Following hybridization, allelic specificity is conferred by enzymatic extension. Products are fluorescently stained and visualization of the resulting signal and decoding of SNP position is performed using the Beadstation scanner and data collection software. Data was analyzed using GenCall v6.2.0.4 and raw genotype files produced with GTS reports v5.1.2.0 (both from Illumina Inc.). Raw genotype data was manipulated and stored in GERON Genotyping (http://neurogenetics.nia.nih.gov), an intranet genotype data repository designed to handle SNP genotypes produced on the Illumina platform.
Real-time PCR
ABI7900Ht Sequence Detector system (Applied Biosystems, Foster City, CA, USA, www.appliedbiosystems.com) was used to perform real-time PCR to confirm the presence of these structural alterations. Primer and probes were designed using Primer express v 2.0.0 software (Applied Biosystems). TaqMan MGB probe for reference gene encoding ß-globin was labeled with 6-FAM, probes against each target region in chromosomes 7, 11, 13 and 16, as well as those 10 targeting the PARK2 locus, on chromosome 6 were labeled with VIC (Applied Biosystems). Primer and probe sequences are available in Table 3. PCR was carried out with TaqMan Universal PCR Master Mix (Applied Biosystems) using 25 ng of genomic DNA, 900 nmol/L primers and 250 nmol/L probes on a total volume of 20 µl. PCR cycling conditions were 95°C for 10 min, 95°C for 15 s and 60°C for 1 min (40 cycles). The plates contained four to six replicates of each genomic DNA sample, control DNA and a no-template water control. The cycle in the log phase of PCR amplification at which a significant fluorescent threshold was reached (Ct) was used to quantify each amplimer. The dosage of each amplimer relative to the reference gene and normalized control DNA was determined using the 2–Ct method. To be considered valid, the requirements were SD of <0.16 and threshold values reached before 28 cycles.
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A value was considered a heterozygous deletion between 0.3 and 0.6, normal between 0.8 and 1.2 and heterozygous duplication between 1.3 and 1.6. Interestingly, individual ND04946, carrying a heterosomic deletion showed a 2–
Ct value between heterozygous deletion and normal, 0.62 (±0.08 SD).
Data analysis
In an effort to examine individual chromosomes for structural mutation, we used the visualization tool Genome Viewer within Beadstudio v2.2.22 (Illumina Inc.). Two metrics were visualized using this tool, B allele frequency and log R ratio. B allele frequency is the theta value for an individual SNP corrected for cluster position, this gives an estimate of the proportion of times an individual allele at each polymorphism was called A or B; thus an individual homozygous for the B allele would have a score close to 1, an individual homozygous for the A allele a score close to 0 and a score of 0.5 would indicate a heterozygous genotype. Significant deviations from these figures in contiguous SNPs are indicative of an alteration in copy number at a locus. The value log R ratio is the log (base 2) ratio of the observed normalized R value for the SNP divided by the expected normalized R value for the SNPs theta value. Expected R is calculated from the values theta and R, where R is the intensity of dye-labeled molecules that have hybridized to the beads on the array and theta is the ratio of signal at each polymorphism for beads recognizing an A allele to beads recognizing a B allele. Thus, the expected R value for any individual at any typed SNP can be calculated using the observed theta at that SNP compared to known values for theta and R (which have been calculated in a large population of typed individuals). Therefore, the ratio of observed R to expected R in any individual at any SNP gives an indirect measure of the binding efficiency of detected alleles for each polymorphism and thus of genomic copy number. An R above 1 is indicative of an increase in copy number, and values below 1 suggest a deletion. While this metric exhibits a high level of variance for individual SNPs, it does provide a measure of copy number when log R ratio values for numerous contiguous SNPs are visualized. Two individuals examined all plots blinded to each others results. All apparent discrepancies were assessed by a third person.
For both Human-1 and HumanHap300 BeadChips individually, homozygosity was scored based on a simple presence/absence basis; the areas of extended homozygosity were first highlighted by a simple script that searched for contiguous tracks of homozygous genotypes greater than 5 Mb, this was then followed by visual examination to rule out a drop in the log R ratio value across the same region, which would indicate genomic deletion (Fig. 1). Once the Human-1 and HumanHap300 data was merged, we also defined a homozygous track as a continuous track of homozygous genotypes where the track had to be at least 1 Mb in length and contain at least 10 SNPs. A summary of the homozygous tracks found in each sample following this criteria, is displayed in Table S1 of the Supplementary Material.
In order to test whether the extended homozygosity and structural genomic alterations were a result of, or amplified by, the LCL creation and passage process we repeated the genome-wide SNP association described above, in DNA extracted directly from the blood samples initially used for LCL creation. We assessed six out of the 26 samples identified with one or more regions of contiguous homozygosity greater than 5 Mb in size, and all 24 samples that harbor putative genomic deletions or multiplications. Data were analyzed as described above.
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SUPPLEMENTARY MATERIAL |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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Supplementary Material is available at HMG Online.
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ACKNOWLEDGEMENTS |
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We would like to thank the subjects for participating in this research and the submitters for depositing samples at the NINDS Neurogenetics repository. We also wish to thank Paul Boyce of Illumina Inc. for technical support. The samples for this study are derived from the NINDS Neurogenetics repository at Coriell Cell Repositories (http://locus.umdnj.edu/ninds/index.html). Access to the samples and to these data are available from this website. This work was supported by the intramural programs of the National Institute on Aging and the National Institute on Neurological Disorders and Stroke (NINDS), National Institutes of Health, Department of Health and Human Services, as well as by an extramural NINDS contract funding the Coriell Repository.
Conflict of Interest statement. The authors declare that they have no competing financial interests.
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FOOTNOTES |
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The authors wish it to be known that, in their opinion, the first four authors should be regarded as joint First Authors. 
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