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Microarray-based comparative genomic hybridization (aCGH) is a powerful diagnostic tool for the detection of DNA copy number gains and losses associated with chromosome abnormalities, many of which are below the resolution of conventional chromosome analysis. It has been presumed that whole-genome oligonucleotide (oligo) arrays identify more clinically significant copy-number abnormalities than whole-genome bacterial artificial chromosome (BAC) arrays, yet this has not been systematically studied in a clinical diagnostic setting.
To determine the difference in detection rate between similarly designed BAC and oligo arrays, we developed whole-genome BAC and oligonucleotide microarrays and validated them in a side-by-side comparison of 466 consecutive clinical specimens submitted to our laboratory for aCGH. Of the 466 cases studied, 67 (14.3%) had a copy-number imbalance of potential clinical significance detectable by the whole-genome BAC array, and 73 (15.6%) had a copy-number imbalance of potential clinical significance detectable by the whole-genome oligo array. However, because both platforms identified copy number variants of unclear clinical significance, we designed a systematic method for the interpretation of copy number alterations and tested an additional 3,443 cases by BAC array and 3,096 cases by oligo array. Of those cases tested on the BAC array, 17.6% were found to have a copy-number abnormality of potential clinical significance, whereas the detection rate increased to 22.5% for the cases tested by oligo array. In addition, we validated the oligo array for detection of mosaicism and found that it could routinely detect mosaicism at levels of 30% and greater.
Although BAC arrays have faster turnaround times, the increased detection rate of oligo arrays makes them attractive for clinical cytogenetic testing.
Molecular cytogenetic techniques such as array-based comparative genomic hybridization (aCGH) have revolutionized cytogenetic diagnostics and, in turn, the clinical management of patients with developmental delays and multiple congenital anomalies [1,2]. These rapid, high-resolution, and highly accurate techniques have identified numerous previously unrecognized chromosomal syndromes [3-8], refined critical regions for established genetic defects [9], and broadened our view of the "normal" diploid genome [10]. In addition, aCGH has given the clinician a greater appreciation of variability in the clinical presentation of many well-described conditions [11,12] and allowed for the discovery of new conditions with relatively mild phenotypes [13,14]. Furthermore, the application of aCGH has created a paradigm shift in genetics that has moved the description and discovery of genetic conditions from the "phenotype-first" approach, in which patients exhibiting similar clinical features are identified prior to the discovery of an underlying etiology, to a "genotype-first" approach, in which a collection of individuals with similar copy-number imbalances can be examined for common clinical features [15].
Originally, targeted microarrays constructed from bacterial artificial chromosomes (BAC) were developed for the clinical laboratory because of their ability to clearly identify copy number changes in discrete regions of the human genome known to play a role in genetic disease [16]. This "less is more" idea prevailed in the early years of clinical aCGH because the technology was new and proof of principle was required before it could be adopted for more widespread diagnostic use. Furthermore, the identification of copy number alterations of unclear clinical significance was considered undesirable to the diagnostician, the ordering physician, and the patient's family. Recently, the coverage of microarrays has expanded to include more comprehensive coverage of the human genome, leading many to suggest that whole-genome BAC or oligo arrays are the next step in the continued improvement in the detection rate of cytogenetic abnormalities.
It has been presumed that whole-genome oligonucleotide arrays, because they have higher resolutions, would detect more copy number aberrations than whole-genome BAC arrays. However, to our knowledge, there has not been a systematic comparison of these two whole-genome copy number screening technologies in a clinical diagnostic environment. Therefore, to determine which platform is most effective in identifying clinically significant DNA copy number alterations, we designed a whole-genome BAC array and a whole-genome oligo array and compared the results in a blinded study of 466 clinical diagnostic specimens. In addition, we prospectively evaluated 3,443 patients by the whole-genome BAC array and 3,096 patients by the whole-genome oligo array and compared the detection rates of clinically significant abnormalities and those of unclear clinical significance. Finally, we validated our oligo array with 48 cases to determine the level of mosaicism that can be reliably detected and compared that level to our previously published cases analyzed using the BAC array.
Materials and methods
Whole-genome BAC array design and aCGH
We constructed a whole-genome BAC array designed for clinical diagnostic use using >4,600 BAC clones. All clones were validated by FISH prior to inclusion on the array using previously described validation procedures [16]. Contigs of 3-6 overlapping clones were selected to cover 1,543 genetic loci, including >150 known microdeletion/microduplication syndromes and increased density of coverage in the 5-10 Mb surrounding the subtelomeric and pericentromeric regions of the genome. In addition, we placed contigs to cover >500 functionally significant genes such as transcription factors and other genes known to play important roles in development. This coverage also includes genome-wide representation with at least one contig in nearly every chromosomal band at the resolution of an 850-band karyotype. The mean gap size for the whole-genome BAC array is ~1.6 Mb. Microarray manufacturing and aCGH analysis using the whole-genome BAC array were performed as previously described [13]. BAC arrays were analyzed after a dye-swap, two-experiment analysis [16], using sex-mismatched controls. Results were then displayed using custom BAC aCGH analysis software (Genoglyphix™; Signature Genomic Laboratories, Spokane, WA).
Whole-genome Oligonucleotide Array Design and aCGH
Oligonucleotide-based microarray analysis was performed using a custom-designed, 105K-feature whole-genome microarray manufactured by Agilent Technologies (Santa Clara, CA) with one probe every 10 kb in regions of interest--microdeletion/microduplication syndromes, the pericentromeric regions, subtelomeres and genes involved in important developmental pathways--for an average of 50 oligos per clinical locus. In addition, to achieve backbone coverage, we placed a probe, on average, every 35 kb throughout the rest of the genome between the regions of interest. Genomic DNA labeling was performed as described for BAC arrays, whereas array hybridization and washing were performed as specified by the manufacturer (Agilent Technologies). A dye swap was not performed for the oligo arrays, and sex-matched controls were used. Arrays were scanned and analyzed as previously described [17]. Results of aberration calls consisting of five or more consecutive oligos were then displayed using custom oligonucleotide aCGH analysis software (Genoglyphix™; Signature Genomic Laboratories). The use of five consecutive oligos achieved a resolution of 40 kb in the regions of interest and a resolution of 140 kb in the backbone.
Decision Algorithm for Clinically Significant Copy Number Reporting
We developed a decision algorithm for classifying clinically significant copy number alterations, alterations of unclear clinical significance, and alterations of no currently known clinical significance. Alterations that were associated with established chromosomal syndromes, were large and affected a significant amount of gene content, or were part of a complex rearrangement such as an unbalanced translocation, insertion, or marker chromosome were characterized as clinically significant. Alterations with unclear clinical significance were most commonly those which were not currently associated with a syndrome but which affected gene content which may have contributed to the patient's phenotype and those which could not be precisely refined by the BAC array. Alterations were considered to have no known clinical significance if they were small, affected minimal gene content, and/or were present in regions where common copy-number variation was known to occur in the general population. Signature's own Genoglyphix Chromosome Aberration Database (GCAD) was used as a reference to assist in the interpretation of each alteration. GCAD is a database of >11,000 chromosomal abnormalities identified in >9,500 patients out of >40,000 patients evaluated by our laboratory and contains detailed statistics of each observed alteration (breakpoint coordinates, size, gene content, etc.) as well as clinical information pertaining to patient referral.
Fluorescence in situ Hybridization (FISH)
When possible, all copy number alterations detected by microarray analysis were visualized by interphase and/or metaphase FISH using a BAC probe located within the region of gain or loss. FISH was performed as previously described [18].
Patient Clinical Testing
To validate the custom-designed 105k oligo array compared to the whole-genome BAC array, 466 cases were run side-by-side in a platform comparison study. In each case, the clinically validated BAC array results were used for interpretation and reporting. Specimens with known chromosome abnormalities, parental specimens, and prenatal cases were excluded from the analysis.
In addition, we conducted a prospective study of 3,443 consecutive BAC microarray analyses and 3,096 consecutive oligo microarray analyses in our clinical laboratory. The array platform used for testing in each case was chosen by the referring physician at the time of sample submission to our clinical diagnostic laboratory. Cases with previously known chromosomal abnormalities, parental samples, and prenatal specimens were again excluded from the data collection.
Mosaicism Assessment
The ability of the oligo platform to detect mosaicism was assessed on 48 patients previously known to carry mosaic abnormalities at levels as low as 5%. The alterations studied included a variety of interstitial, terminal, and whole-chromosome copy-number abnormalities, as well as marker chromosomes. The mosaic alteration in each patient was initially assessed by BAC array and the level of mosaicism determined by interphase FISH analysis when possible. In a separate experiment, mosaicism was assessed using a dilution of cells from a male with trisomy 21 with normal male control cells, as previously described [19]. After FISH verification of the dilutions, DNA was extracted from the diluted cells, labeled and hybridized to the custom-designed oligo array as described above.
Platform comparison study
From the 466 cases analyzed by the BAC array, using the previously described algorithm, we excluded 347 cases that only had aberrations located within regions that contained no genes and/or aberrations that had been established to be normal population variants by Signature Genomic Laboratories or identified in the Toronto Database of Genomic Variants (DGV, http://projects.tcag.ca/variation/ webcite). After these cases were excluded, 138 copy number alterations in 119 cases (25.5% of the original 466 cases) remained that required FISH analysis. These aberrations included subtelomeric and pericentromeric gains for which FISH was required to exclude an unbalanced translocation or a marker chromosome. After FISH was performed, 60 aberrations in 52 cases were classified as normal variants because marker chromosomes and derivative chromosomes were not identified and because these alterations were located within regions where common copy number variation is known to occur. Thus, alterations of potential clinical significance according to our algorithm were identified in 67 cases, a detection rate of 14.4%. Of these cases, 56 (12.0%) were considered to contain clinically significant copy number alterations (Table 1), and 11 (2.4%) were considered to contain copy number variants of unclear clinical significance for which parental analyses were recommended to further clarify the abnormality (Table 2). aCGH and FISH analysis performed on parental samples revealed that six alterations of unclear significance were inherited from a carrier parent and one was a de novo event in the proband. The origin of the other four unclear alterations could not be determined.
Table 1. Cases with Alterations of Clinical Significance Identified by BAC and Oligo aCGH.
Table 2. Cases with Alterations of Unclear Significance Identified by BAC and Oligo aCGH.
Using the oligo array, we identified 1,337 copy number variations among the same 466 cases. Using the algorithm previously described, we excluded 1,172 aberrations that were located within regions that had no gene content or those that were common copy number variants. After these exclusions were made, 165 aberrations in 138 cases (29.6%) remained that required FISH analysis. After FISH analysis was performed, aberrations of potential clinical significance were identified in 73 cases, a detection rate of 15.7%. Of these, the same 56 (12.0%) cases that were identified by the BAC platform were considered to contain clinically significant alterations (Table 1) and 17 (3.7%) were determined to contain copy number variants of unclear clinical significance.
Table 3 shows the six cases for which aberrations of unclear clinical significance were identified by the oligo array but not by the BAC array. In all six cases, the aberrations either fell within the gaps in the BAC array coverage or were only partially covered by one or more BACs. The average size of the alterations that were not detected by the BAC array was 1.12 Mb (range: 289 kb - 1.42 Mb).
Table 3. Cases with Alterations of Unclear Significance Detected by Oligo Array but not Identified by BAC Array.
In two cases, the oligo microarray identified additional complexity that was not recognized by the BAC array. In patient 21566, the BAC array identified one interstitial deletion of 17p13.2p13.1, whereas oligo array analysis identified that deletion and an additional interstitial deletion in the same band (data not shown). In patient 21897, the BAC array identified a 6.8 Mb terminal deletion of 5p, whereas oligo array analysis identified that deletion and a 1.4 Mb duplication proximal to the deleted region (Figure 1).
Figure 1. Identification by oligonucleotide microarray of additional complexity missed by BAC microarray. (A) BAC microarray results showing a single-copy loss of 34 BAC clones from the terminus of 5p, approximately 6.8 Mb in size (chr5: 387,034-7,150,950, based on UCSC 2006 hg 18 assembly). Probes are ordered on the x axis according to physical mapping positions, with the p-arm probes to the left and q-arm probes to the right. (B) shows oligonucleotide microarray results of the terminal deletion shown in (A) in addition to single-copy gain of 29 probes from 5p, approximately 1.38 Mb in size (chr5: 8,511,592-9,888,817, based on UCSC 2006 hg 18 assembly). Probes are ordered as in the BAC array. Regions shaded in blue represent deletions detected by microarray, whereas duplications are shaded in pink.
Prospective Diagnostic Comparison
Of the 3,443 diagnostic specimens analyzed using our whole-genome BAC array, 605 (17.6%) had copy number alterations. Using the previously described algorithm, 365 (10.6%) had abnormalities that were classified as clinically significant, whereas 240 (6.9%) had copy number variants of unclear clinical significance.
Of the 3,096 diagnostic specimens analyzed using our whole-genome oligo array during the same time period, 698 (22.5%) had copy number alterations. Using the previously described algorithm, 477 (15.9%) of these cases were determined to contain alterations considered to be clinically significant and 221 (7.0%) were determined to contain copy number variants of unclear clinical significance (Table 4).
Table 4. Summary of the Prospective Diagnostic Comparison.
The increased number of cases with clinically significant alterations detected by the oligo array was found to be statistically significant using a Fisher's Exact Test (OR = 1.5359, p < .0001). The increased number of cases with alterations of unclear significance detected by the oligo array was not statistically significant (OR = 1.0259, p = 0.8090).
Mosaicism Assessment
All but three of the 48 previously known mosaic alterations were detected by the oligo array. FISH analysis estimated that the proportion of uncultured cells carrying the alteration was 24% in the first case, while the proportion in cultured cells was 6%. In the second case, 5% of cells were found to carry the alteration by FISH (data not shown). The proportion of cells carrying the alteration in the third case could not be determined because FISH confirmation was not possible on the sample received by our laboratory. Certain alterations, such as tetrasomy 12p, were successfully detected in proportions of cells as low as 10% by the oligo array, although this low threshold of detection was facilitated by the tetrasomic nature of the rearrangement; the 4:2 ratio of patient to control DNA in this case was more readily detected than the 3:2 ratio typically associated with duplications. Figure 2 shows a 2.77 Mb interstitial deletion at 16q12.1 present in 23% of cultured metaphase cells that was detected by the oligo array.
Figure 2. Oligonucleotide microarray analysis of a mosaic 16q12.1 deletion (shaded blue region). The zoomed-in microarray plot shows a single-copy loss of 289 probes from 16q12.1, approximately 2.77 Mb in size (chr16: 46,837,260-49,605,054, based on UCSC 2006 hg 18 assembly). Probes are ordered on the x axis according to physical mapping positions, with the most proximal 16q11.2 probes to the left and the most distal 16q12.2 probes to the right.
In the dilution series of trisomy 21 cells, shifts in the aCGH data were distinguishable down to levels as low as 10%, but could only be readily detected at a level of 30% or greater (Figure 3). As the proportion of trisomy 21 cells was increased from 10% to 30%, the average log2 ratio of chromosome 21 increased from 0.08 to 0.21. During the prospective diagnostic comparison, 16 cases analyzed using the BAC array contained mosaic alterations, whereas only 12 mosaic cases were identified using the oligo array (Table 5). The increased number of mosaic abnormalities detected by the BAC array was determined to be not statistically significant (OR = 1.1999, p = 0.7066).
Figure 3. Oligonucleotide microarray analysis of artificially derived mosaic trisomy 21 samples. (A) 10% trisomy 21 showing a very subtle copy-number gain for all clones on chromosome 21. The profile was generated using DNA extracted from a mixture of blood which contained 10% WBCs from a trisomy 21 subject and 90% WBCs from a normal male individual. (B) 15% trisomy 21, generated as in (A), showing a very subtle copy-number gain for all clones on chromosome 21. (C) 20% trisomy 21, generated as in (A) showing a subtle copy-number gain for all clones on chromosome 21. (D) 30% trisomy 21, generated as in (A), showing a clear copy-number gain for all clones on chromosome 21. The inset images to the right of each array plot show the average log2 ratio of all probes mapping to chromosome 21, with the horizontal dotted line representing a log2 ratio of zero and the vertical dotted line representing the centromere. A pink bar plotted above the horizontal line represents a copy-number gain of all probes on chromosome 21. To the left of each inset image is the average log2 ratio at the specified proportion of trisomic cells.
Table 5. Mosaic Alterations Detected in the Prospective Diagnostic Comparison.
BAC and oligo array platforms each have unique advantages and disadvantages in a diagnostic setting; these may include turnaround times, genomic coverage, and costs. One of the most important characteristics of each platform is the detection rate of clinically significant alterations. Our results demonstrate that our whole-genome oligo array was able to detect such alterations in 15.4% of patients tested, compared to the BAC array detection rate of 10.6%, a statistically significant difference (Fisher's Exact Test, p < 0.0001). The alterations that constitute the 4.8% difference in detection rate between the BAC and oligo arrays are either too small to be detected by the BAC array but are not below the resolution of the oligo platform (Figure 4) or fall within gaps in the BAC array coverage (Figure 5). Figure 4 shows a 44 kb deletion of 17p13.3 detected in a patient referred to our laboratory for convulsions. This deletion encompasses the first exon of PAFAH1B1 (LIS1). While it is not known whether this deletion results in a null allele or simply a truncated gene product, hemizygous deletions and mutations of this gene are found in patients with isolated lissencephaly type 1 (OMIM 607432) and have been linked to epileptic seizures and convulsions [20,21]. Although RP11-135N5 provides coverage of this region on the BAC array, FISH analysis using this clone could not confirm the deletion in any cells because of the deletion's small size compared to the FISH probe used. Thus, this clinically significant deletion could only have been reliably detected using the oligo platform. Although oligo-based aCGH has the power to detect alterations smaller than the size of a BAC probe, BAC-based aCGH has an advantage in that the analysis makes evident the appropriate probe to be used for FISH confirmation. In addition, this probe is usually readily available because of its inclusion on the microarray platform and will have a high rate of successful confirmation. When oligonucleotide-based aCGH is performed, BAC probes must be specifically selected for the FISH confirmation of each small abnormality that is detected. Once a probe has been selected, it must also be specially prepared or ordered before FISH can be performed. This process increases both the time it takes to perform FISH confirmation of oligo aCGH results and the cost associated with the analysis.
Figure 4. Oligonucleotide microarray characterization of an interstitial deletion at 17p13.3. The zoomed-in microarray plot shows a single-copy loss of six probes from the short arm of chromosome 17 at 17p13.3, approximately 44.0 kb in size (chr17: 2,415,074-2,459,051, based on UCSC 2006 hg 18 assembly). Probes are ordered on the x axis according to physical mapping positions, with the most distal 17p13.3 probes to the left and the most proximal 17p13.3 probes to the right. Below is a schematic of the deletion region. The deletion disrupts the PAFAH1B1/LIS1 gene.
Figure 5. Oligonucleotide microarray characterization of an interstitial deletion at 6q14.1. The zoomed-in microarray plot shows a single-copy loss of 43 oligonucleotide probes from the long arm of chromosome 6 at 6q14.1, approximately 2.9 Mb in size (chr6: 79,838,518-82,730,466, based on UCSC 2006 hg 18 assembly). Probes are ordered on the x axis according to physical mapping positions, with the most proximal 6q14.1 probes to the left and the most distal 6q14.1 probes to the right. Below is a schematic of the deletion region. Blue and gray boxes represent genes in the deletion region.
Figure 5 shows a 2.9 Mb deletion of 6q14.1 detected in a patient referred to our laboratory for developmental delay and dysmorphic features. This deletion encompasses eight genes: PHIP, HMGN3, LCA5, SH3BGRL2, ELOVL4, TTK, BCKDHB, two of which are known to be associated with human disease [22-24]. Although this 2.9 Mb deletion is likely to be clinically significant, it lies within a gap in the coverage of our BAC array and could only be detected using the oligo platform because of its more uniform backbone coverage.
The detection rate of alterations of unclear clinical significance is also a concern during the selection of a microarray platform in a clinical diagnostic setting. Our data suggest that both the oligo and BAC platforms detect similar numbers of abnormalities of unclear significance (7.0% by BAC and 7.1% by oligo), although the circumstances leading to an unclear clinical interpretation may vary between the platforms. On the BAC platform, unclear results are often associated with gaps in coverage which prevent the precise determination of the breakpoints and gene content of an abnormal region. This lack of information prohibits definitive interpretation of the clinical significance of the alteration. Figure 6 presents a 262 kb deletion of 9q33.1 detected by BAC array in a patient referred for developmental delay, dysmorphic features, and multiple congenital anomalies. The boundaries of this alteration as defined by BAC array include only one gene, TLR4 [25]. However, gaps in BAC coverage on both sides of the alteration span 4.5 Mb proximally and 4.0 Mb distally. As a result of these coverage gaps, this alteration, though estimated to be just 262 kb, may be as large as 8.7 Mb and include up to 48 additional genes. The design of BAC arrays with dense clone coverage is possible; however, probe density is limited by the availability of BAC clones and the presence of potentially interfering genomic architecture such as segmental duplications. In addition, BAC-based microarrays will not reliably detect abnormalities smaller than the size of an individual probe--80-200 kb, on average, for BAC clones.
Figure 6. BAC microarray characterization of a 9q33.1 deletion. The zoomed-in microarray plot shows a single-copy loss of three BAC clones from the long arm of chromosome 9 at 9q33.1, approximately 262 kb in size (chr9: 119,452,279-119,714,054 based on UCSC 2006 hg 18 assembly). The nearest distal clone on chromosome 9 that is not deleted is RP11-977E8 and is approximately 4.0 Mb away from the deleted region. The nearest proximal clone on chromosome 9 that is not deleted is RP11-999I23 and is approximately 4.4 Mb away from the deleted region. Probes are ordered on the x axis according to physical mapping positions, with proximal 9q32 clones to the left and distal 9q33.2 clones to the right. Below is a schematic of the deletion region. Vertical blue lines represent the minimum size of this alteration, which encompasses one gene, TLR4.
Although gaps in coverage and limited breakpoint-resolving power are primarily a concern for BAC platforms, both oligo and BAC platforms produce results that are unclear because a lack of published evidence prevents a conclusive association between the gene content of an alteration and the clinical features of the patient from being made. Figure 7 presents a 160 kb deletion of 4q25 detected by oligo array in a patient referred for developmental delay. Follow-up analysis performed on this patient's parents revealed that this alteration was de novo in origin. This alteration deletes two genes, PAPSS1 and SGMS2. While mutations or alterations of these genes have not been associated with disease in humans, it has been shown that PAPSS1 plays a key role in post-translational modification and SGMS2 mediates the production of sphingomyelin [26,27]. Thus, although the gene content and inheritance pattern of this deletion suggest a causative role in the patient's clinical features, a lack of published information linking the genes affected by this alteration with a distinct phenotype prevents a clear interpretation from being made based on only aCGH results. This type of unclear result, although more prominent with oligo platforms (4.2% by BAC vs. 7.1% by oligo), is an element of all aCGH analysis regardless of platform and accentuates the need for databases containing aCGH results in combination with phenotypic information. Although the number of characterized genetic disorders and genomic regions is rapidly increasing, the clinical consequences of alterations involving much of the genome still remain unclear.
Figure 7. Oligonucleotide microarray characterization of an interstitial deletion at 4q25. The zoomed-in microarray plots shows a single-copy loss of 15 oligonucleotide probes from the long arm of chromosome 4 at 4q25, approximately 159.6 kb in size (chr4: 108,834,399-108,994,048, based on UCSC 2006 hg 18 assembly). Probes are ordered on the x axis according to physical mapping positions, with proximal 4q25 clones to the left and distal 4q25 clones to the right. Below is a schematic of the deletion region. The deletion disrupts the PAPSS1 and SGMS2 genes, represented by blue boxes.
The increase in the number of copy number alterations identified by higher-resolution whole-genome arrays underscores the need for a variety of tools to facilitate the interpretation of array results in a clinical diagnostic setting. We propose the use of an algorithm such as the one outlined here in conjunction with databases of normal population variants, clinically significant alterations, and those of unclear significance. Although such databases can provide invaluable context for the analysis of aCGH data, care must be taken by the diagnostician when comparing their data to pre-existing databases of copy-number variations. For example, data in the DGV are pooled from a variety of sources, platforms, and populations using a variety of different controls and without independent verification, and thus may not be appropriate for comparison in all situations. Furthermore, recent evidence suggests that most data in the DGV overestimate the size of the regions involved because they are dependent primarily on BAC array data, which has a tendency to overestimate the true size of small aberrations [28]. Thus, the most useful CNV databases may be those generated by individual laboratories using identical reference controls and array platforms. Based on our experience, we have constructed a database of abnormal copy number aberrations identified by BAC and oligo aCGH in our laboratory and a database of copy-number variations thought to have no significance. Such databases are essential for understanding the various copy number aberrations identified by microarray analysis.
Genotype-phenotype correlations in a diagnostic setting must address a variety of factors including gene content, potential position effects, aberration size, and inheritance patterns. These factors often present conflicting evidence about the potential clinical significance of a rare alteration. For instance, the size of an abnormality is commonly used as justification for its proposed clinical consequences; however, this association is not always straightforward. High-resolution microarray analysis routinely detects abnormalities smaller than 500 kb that disrupt clinically significant genes and have clear phenotypic impact (Figure 4); conversely, numerous examples of common copy-number variants have been observed that are relatively large but lie in regions with sparse gene content. In addition, although it is generally assumed that de novo abnormalities are causative and inherited abnormalities are not, this is not always the case. There are a number of regions of the genome where both inherited and de novo copy number alterations have been identified, some of which result in mild phenotypes that may be inherited from parents who have a milder or subclinical, presentation. For example, deletions of distinct regions of 1q21 have been associated with both thrombocytopenia absent radius (TAR) syndrome and a variable phenotype including microcephaly/macrocephaly, developmental delay, cardiac abnormalities, and schizophrenia [29-31], but in many instances aberrations of these regions are inherited from phenotypically normal parents [32]. Another example is the 16p11.2 region associated with a range of cognitive, developmental, and speech delays, behavioral issues, and autism, deletions and duplications of which can be inherited or de novo [33-36]. In regions such as these, copy number changes may unmask recessive alleles or work in conjunction with various genetic modifiers, perhaps even other CNVs, to produce a clinical phenotype. Potentially, non-paternity may also confound genotype-phenotype correlation for copy number alterations in these complex regions of the genome. These reasons underscore the need for thorough databases of normal population variants and clinically significant alterations complete with genotype-phenotype correlations. Such databases expedite the process of determining the potential significance of copy-number alterations in a diagnostic setting; aid in the elucidation of new microdeletion/duplication syndromes and new regions of benign copy-number variation; and help reduce the burden of expensive, time-consuming, and difficult follow-up necessitated by the increased number of alterations of unclear clinical significance detected by microarray analysis.
We [19] and others [37] have shown that mosaicism can be detected at low frequencies of chromosomally abnormal cells using BAC-based aCGH; however, the ability of oligo platforms to reliably detect mosaic abnormalities has not yet been well established. Our current assessments demonstrate that aCGH using either BAC or oligo platforms can easily detect mosaicism of 30% or greater for a variety of alterations and that levels as low as 10% can be detected with both platforms under optimal conditions. In addition, our retrospective analysis showed that there is no significant difference between the two types of platforms in the number of mosaic abnormalities detected in a clinical diagnostic setting (p = 0.7066). However, BAC-based arrays may still have a greater ability to detect mosaic abnormalities present at very low levels (less than 20%), perhaps due to the routine use of dye-swap experiments which can be cost-prohibitive with oligo arrays but promote the visual identification of mosaic abnormalities. The sensitivity of the BAC array is demonstrated by the detection in three cases of abnormalities in only 10% of cells during the retrospective study, whereas the lowest level of mosaicism detected by our oligo array was 21% (Table 5). The ability of an aCGH platform to detect mosaic abnormalities also depends largely on the effectiveness of the software used to analyze the data, as low-level mosaic alterations are difficult to identify using only visual inspection (Figure 3). For this reason, it is important to select analysis software which facilitates the identification of mosaic alterations.
These data suggest high-resolution oligo-based aCGH detects a higher proportion of clinically significant abnormalities than BAC-based aCGH. Our results also demonstrate the ability of microarray-based CGH to reliably produce high-yield results in a clinical setting using differing platforms, array designs, and analysis algorithms, supporting the validity of array CGH as a first-tier diagnostic screening tool [38]. Finally, the prevalence of copy number variants of unclear clinical significance detected on both platforms underscores the need for the development of readily accessible diagnostic tools in the form of databases of documented chromosome abnormalities to aid in the interpretation of microarray data.
Competing interests
NN, BST, BCB, BAB and LGS are employees of Signature Genomic Laboratories, a subsidiary of PerkinElmer.
Authors' contributions
NN wrote the manuscript; BST analyzed the molecular cytogenetics results; BAB, BCB and LGS designed and coordinated the study. All authors have read and approved the final manuscript.
We thank Aaron Theisen (Signature Genomic Laboratories, Spokane, WA) for his careful edits of the paper and all of the laboratory staff at Signature Genomics for conducting the aCGH and FISH experiments.
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Nat Genet 2004, 36:955-957. PubMed Abstract | Publisher Full Text
Sharp AJ: Emerging themes and new challenges in defining the role of structural variation in human disease.
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Curr Opin Pediatr 2005, 17:740-746. PubMed Abstract | Publisher Full Text
Gropman AL, Elsea S, Duncan WC Jr, Smith AC: New developments in Smith-Magenis syndrome (del 17p11.2).
Curr Opin Neurol 2007, 20:125-134. PubMed Abstract | Publisher Full Text
Ballif BC, Theisen A, Coppinger J, Gowans GC, Hersh JH, Madan-Khetarpal S, Schmidt KR, Tervo R, Escobar LF, Friedrich CA, McDonald M, Campbell L, Ming JE, Zackai EH, Bejjani BA, Shaffer LG: Expanding the clinical phenotype of the 3q29 microdeletion syndrome and characterization of the reciprocal microduplication.
Mol Cytogenet 2008, 1:8. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Coppinger J, McDonald-McGinn D, Zackai E, Shane K, Atkin JF, Asamoah A, Leland R, Weaver DD, Lansky-Shafer S, Schmidt K, Feldman H, Cohen W, Phalin J, Powell B, Ballif BC, Theisen A, Geiger E, Haldeman-Englert C, Shaikh TH, Saitta S, Bejjani BA, Shaffer LG: Identification of familial and de novo microduplications of 22q11.21-q11.23 distal to the 22q11.21 microdeletion syndrome region.
Hum Mol Genet 2009, 18:1377-1383. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Shaffer LG, Theisen A, Bejjani BA, Ballif BC, Aylsworth AS, Lim C, McDonald M, Ellison JW, Kostiner D, Saitta S, Shaikh T: The discovery of microdeletion syndromes in the post-genomic era: review of the methodology and characterization of a new 1q41q42 microdeletion syndrome.
Genet Med 2007, 9:607-616. PubMed Abstract | Publisher Full Text
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Am J Med Genet A 2006, 140:2757-2767. PubMed Abstract | Publisher Full Text
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Brunetti-Pierri N, Berg JS, Scaglia F, Belmont J, Bacino CA, Sahoo T, Lalani SR, Graham B, Lee B, Shinawi M, Shen J, Kang SH, Pursley A, Lotze T, Kennedy G, Lansky-Shafer S, Weaver C, Roeder ER, Grebe TA, Arnold GL, Hutchison T, Reimschisel T, Amato S, Geragthy MT, Innis JW, Obersztyn E, Nowakowska B, Rosengren SS, Bader PI, Grange DK, et al.: Recurrent reciprocal 1q21.1 deletions and duplications associated with microcephaly or macrocephaly and developmental and behavioral abnormalities.
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Stefansson H, Rujescu D, Cichon S, Pietilainen OP, Ingason A, Steinberg S, Fossdal R, Sigurdsson E, Sigmundsson T, Buizer-Voskamp JE, Hansen T, Jakobsen KD, Muglia P, Francks C, Matthews PM, Gylfason A, Halldorsson BV, Gudbjartsson D, Thorgeirsson TE, Sigurdsson A, Jonasdottir A, Bjornsson A, Mattiasdottir S, Blondal T, Haraldsson M, Magnusdottir BB, Giegling I, Moller HJ, Hartmann A, Shianna KV, et al.: Large recurrent microdeletions associated with schizophrenia.
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Klopocki E, Schulze H, Strauss G, Ott CE, Hall J, Trotier F, Fleischhauer S, Greenhalgh L, Newbury-Ecob RA, Neumann LM, Habenicht R, Konig R, Seemanova E, Megarbane A, Ropers HH, Ullmann R, Horn D, Mundlos S: Complex inheritance pattern resembling autosomal recessive inheritance involving a microdeletion in thrombocytopenia-absent radius syndrome.
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Mefford HC, Sharp AJ, Baker C, Itsara A, Jiang Z, Buysse K, Huang S, Maloney VK, Crolla JA, Baralle D, Collins A, Mercer C, Norga K, de Ravel T, Devriendt K, Bongers EM, de Leeuw N, Reardon W, Gimelli S, Bena F, Hennekam RC, Male A, Gaunt L, Clayton-Smith J, Simonic I, Park SM, Mehta SG, Nik-Zainal S, Woods CG, Firth HV, et al.: Recurrent rearrangements of chromosome 1q21.1 and variable pediatric phenotypes.
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Kumar RA, KaraMohamed S, Sudi J, Conrad DF, Brune C, Badner JA, Gilliam TC, Nowak NJ, Cook EH Jr, Dobyns WB, Christian SL: Recurrent 16p11.2 microdeletions in autism.
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Marshall CR, Noor A, Vincent JB, Lionel AC, Feuk L, Skaug J, Shago M, Moessner R, Pinto D, Ren Y, Thiruvahindrapduram B, Fiebig A, Schreiber S, Friedman J, Ketelaars CE, Vos YJ, Ficicioglu C, Kirkpatrick S, Nicolson R, Sloman L, Summers A, Gibbons CA, Teebi A, Chitayat D, Weksberg R, Thompson A, Vardy C, Crosbie V, Luscombe S, Baatjes R, et al.: Structural variation of chromosomes in autism spectrum disorder.
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Miller DT, Adam MP, Aradhya S, Biesecker LG, Brothman AR, Carter NP, Church DM, Crolla JA, Eichler EE, Epstein CJ, Faucett WA, Feuk L, Friedman JM, Hamosh A, Jackson L, Kaminsky EB, Kok K, Krantz ID, Kuhn RM, Lee C, Ostell JM, Rosenberg C, Scherer SW, Spinner NB, Stavropoulos DJ, Tepperberg JH, Thorland EC, Vermeesch JR, Waggoner DJ, Watson MS, et al.: Consensus statement: chromosomal microarray is a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies.
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text:
Follow GO:
At Metrolinx, we are committed to completing the Georgetown South (GTS) Project with minimal disruption to our neighbours. That is why we have developed a Noise and Vibration Mitigation and Monitoring Strategy for construction and operation of the GTS Project.
The strategy builds on the noise and vibration recommendations from the project's Environmental Assessment study, and provides an overview of the general mitigation measures for the project. It is also the basis for site-specific construction noise and vibration mitigation and monitoring plans prepared for individual segments of the rail corridor during construction, and operational noise and vibration assessments.
We will review major construction sites prior to construction and monitor them throughout all construction phases for both noise and vibration levels. If levels are found to be above typical levels for construction for sustained periods of time, we will investigate the source of the noise and implement mitigation measures as required.
To view the specific reports for each project, please see the Noise and Vibration section on the following pages:
Black Creek/Black Creek Drive |
Drive Underpass | Corridor Grading, Track
and Signals | Denison Road Underpass | Humber River Bridge | Strachan Avenue Overpass | Weston Tunnel | West Toronto Diamond Grade Separation
The majority of the work will take place during regular construction hours (Monday to Friday, 7 a.m. to 7 p.m.) and Saturdays (9 a.m. to 7 p.m.) when necessary. This respects the City of Toronto noise by-law.
However, because of certain restrictions, we may be required to work outside the regular construction hours. One of the biggest challenges we face is that we are working on an active corridor. This means that we are restricted in the work we can do during peak times due to train movement.
We will provide advance notice to the surrounding community when these extended hours are planned.
There are several factors we will consider in assessing noise during construction. This includes identifying sensitive receptors. Sensitive receptors are areas that are considered to be sensitive to noise, including residential dwellings, schools, daycare facilities, hospitals, seniors' residences, and places of worship. Commercial and industrial buildings are typically not considered sensitive receptors relative to noise.
We will consider the existing noise environment, the noise levels during construction activities and the adjacent land use. We will also assess the potential noise effects from haul roads for construction-related trucks, based on projected haul traffic counts, including dump truck and delivery truck movements and excavation haul traffic volumes.
We want to be proactive in mitigating noise from the construction project where required. That is why we are assessing several factors that will allow us to determine what, if any, mitigation will be required during construction and for how long it will be needed. These factors include:
We will take several actions in an effort to mitigate noise during construction of this project. Metrolinx will provide a detailed impact assessment and comply with the Ministry of Environment operating standards. All construction equipment on site will be operated in accordance with Ministry of the Environment publication NPC-115.
We will use vehicles and equipment with efficient muffling devices and employ alternative equipment that generates less noise where possible. This includes avoiding impact pile driving where possible. We will install rubber linings in equipment such as chutes and dumpers to reduce impact noise.
Vehicles that are used for extended periods of time (two days or more) will be fitted with sound reducing back up (reversing) alarms, while adhering to construction safety standards. We will also supply electrical power directly from the grid where possible to avoid continuous use of generators.
In addition to these mitigation measures, specific areas along the corridor may require supplementary mitigation, depending on the noise generated and duration of construction activities. In these cases, supplementary mitigation that may be implemented includes:
Metrolinx is working directly with independent third parties to regularly monitor construction noise along the corridor.
During construction, we will conduct noise monitoring at locations that are representative of the nearest sensitive receptors.
To monitor the levels, we will take into account the existing noise environment, the absolute noise levels during construction activities, the duration of the construction and the adjacent land use.
If levels are determined to be above typical noise levels for construction activity, and have a long duration, the noise monitoring reports will be reviewed and, if necessary, additional measurements will be done at the location of concern to determine if further mitigation is needed.
Where construction work unrelated to this project is underway in the same area, there is a chance increased noise and vibration generated by other construction activities outside the Georgetown South Project may impact noise and vibration levels in the community. In such cases, the owners of those unrelated projects are responsible for managing the noise and vibration resulting from construction on their sites.
Prior to construction, we will conduct pre-construction condition inspections for all buildings within
50-100m of the work area. This inspection documents the current condition of the property prior to construction, and involves taking photographs outside and inside the property. It also includes the inspection and reporting on pre-construction foundation and structural conditions of any historic resources in the area, including designated heritage buildings and structures.
During construction, we will assess vibration with respect to human perceptibility and potential building damage. We will measure the baseline vibration levels to benchmark current vibration levels, so we can compare levels during construction with the baseline to determine the level generated by our construction.
We will investigate impacts where in-ground vibration levels are 5 mm/s (millimeters per second) or more.
We will undertake several measures to mitigate vibration during construction of the project. This includes:
In addition to these measures, specific areas along the corridor may require supplementary mitigation, depending on the vibration generated and the duration of construction activities. This includes:
Metrolinx will work directly with independent third parties to regularly monitor construction vibration along the rail corridor. We will conduct vibration monitoring at locations representative of the nearest sensitive locations.
In the event that the vibration measurements are beyond regular construction levels and have a long duration, the reports will be reviewed and, if necessary, additional measurements will be done at the location of concern to determine if further mitigation is needed.
Where construction work unrelated to this project is underway in the same area, there is a chance increased noise and vibration generated by other construction activities outside the GTS Project may impact noise and vibration levels in the community. In such cases, the owners of those unrelated projects are responsible for managing the noise and vibration resulting from construction on their sites.
We have developed a Noise and Vibration Mitigation and Monitoring Strategy to address both the construction work over the next few year as well as operation of our expanded rail service to minimize the impact to our surrounding communities.
Operational Noise and Vibration Mitigation Report
The Operational Noise and Vibration Mitigation report provides recommendations to address operational noise and vibration impacts that will occur after enhanced train service is introduced in the Georgetown South rail corridor over the next 15 years. Noise mitigation is required when there is an impact of 5 dB or greater and vibration mitigation when there is a 25% increase above 0.14 mm/s RMS.
Main Report
Appendix A – Community Feedback
Appendix B – Train Volume and Speed Projections
Appendix C – Detailed Results of Operational Noise Impact Assessment
Appendix D – Planned Noise Mitigation
Appendix E – Example Noise Wall Images
Appendix F – Planned Vibration Mitigation
The report recommends noise mitigation that is required for the service levels planned for 2015 as well as for Future Build service levels (i.e. the long-term plan for
full-day, two-way service). Based on the community's feedback, Metrolinx has decided to install the Full Build noise mitigation (sound walls) for Opening Day 2015 along the Georgetown South Project rail corridor. As a result, noise levels will be quieter than they are today.
Based on an additional review of the modelled results, we will be providing noise walls at all locations recommended in the 2009 Environmental Project Report as well as some additional places where we believe noise walls are merited because of unique situations.
Thank you to everyone who attended our operational noise and vibration mitigation public meetings in November 2011 and our public meetings in February 2012.
Continuing Consultation
Thank you to everyone who expressed an interest in participating in our Noise
Wall Community Advisory Committees (CACs). To help guide the design of noise
walls in each neighbourhood, eight Committees were established along the
corridor, from Bathurst Street to the Humber River Bridge. Each Committee had a
mix of local residents and community groups. The CACs provided an
opportunity for local neighbourhoods to help design the look and feel of the
walls that will reduce sound as train service increases along the corridor.
Learn more ...
Operational Noise and Vibration Study, finalized in February 2012, outlined
operational noise and vibration mitigation along the Georgetown South rail
corridor. No vibration mitigation was required between Rogers Road and Jane
Street because the proposed new tracks followed the existing track alignment
with no projected train speed increases.
However, in April 2013, the tracks were realigned to protect for a future
Eglinton GO Station. Track 4, the western most track to be added to the
corridor, will be aligned closer to the west side of the corridor from
approximately Ray Avenue to Cobalt Street. The two tracks being added for 2015
now require vibration mitigation.
At a public meeting at the Mount Dennis Legion on June 26, 2013, these changes in
vibration mitigation were shared with the community. An additional commitment
was made to provide vibration mitigation for the addition of a future fourth
track and the eventual replacement of the existing track.
Presentation |
Updated Aerials
Since the West Toronto Diamond Grade Separation had its own environmental
assessment process and did not address noise impacts related to the Georgetown
South Project, we decided to complete a separate noise and vibration study for
this section. The noise walls for the West Toronto Diamond will be installed at
the same time as the rest of the Georgetown South Project.
The West Toronto Diamond Grade Separation Noise and Vibration Study is now
complete. The results show that no vibration mitigation is required and only a
small number of noise walls are needed between the project limits of St. Clair
St. W. and Dupont St. This is because the construction of the West Toronto
Diamond Grade Separation will lower the GO rail corridor which significantly
reduces train noise.
West Toronto Diamond – Operational
Noise and Vibration Assessment
We will include this section of noise wall consultation with the existing noise
wall community advisory committee covering the area between Bloor St. W. and
Dupont St. We believe that since this section includes part of the West Toronto Railpath,
there is an opportunity to ensure consistency in design between the areas. We
will be engaging with the community to ensure that it stays up to
date on the design process.
If you have any questions, please do not hesitate to contact 416-604-9582 or
In response to community questions about whether the noise walls would still
be required if the Union Pearson (UP) Express and GO service was electrified,
Metrolinx completed an assessment to identify exactly what the noise levels
would be with an electrified service. Georgetown South Rail Corridor Expansion –
Assessment of Noise Impacts with Electrification of Union Pearson Express and GO
Trains has now been
The results of this study demonstrate that the average noise level difference
between an electric-powered and diesel-powered rail service for both the UP
Express and GO service is negligible. The average decrease in noise is about 2
decibels. Please see the full report below to review the electrification noise
An electrified rail service for both UP Express and GO Transit still requires
noise walls for the vast majority of locations. For the few locations that would
no longer have a 5 decibel increase with electrification of the future train
service levels, GO Transit has determined that the noise increases would still
be significant and local communities would benefit from the installation of a
noise wall.
Georgetown South
Rail Corridor Expansion – Assessment of Noise Impacts with Electrification of
Union Pearson Express and GO Trains
If you have any concerns about noise and vibration related to the Georgetown South Project, please contact us at email@example.com.
Alternatively, you can visit or call our Strachan or Weston Community Office. The Community Relations team at these offices will take a report of your questions or concerns and will work with the project delivery team to investigate.
Sign up to receive email updates on project information. Subscribe now!
Revised: November 27, 2015 | Copyright © GO Transit 2015 | Careers | Site Map | Privacy & Website Terms | Contact Us

subset: commoncrawl


text:
Jogger Rebekah Bletsch was shot to death along Automobile Road in Muskegon County's Dalton Township on June 29, 2014. / WZZM-TV
DALTON TOWNSHIP, MICH. — Reward money is being offered for information regarding the fatal shooting of a Muskegon County jogger whose body was found along the side of a road.
The family of Rebekah Sue Bletsch is offering a $10,000 reward for information leading to the arrest and conviction of those responsible for her death. Silent Observer is also offering $2,500 toward the reward, which is expected to grow.
The 36-year-old wife and mother from Dalton Township was jogging her normal route on Automobile Road Sunday afternoon when someone shot her in the head. Authorities say she had valuables on her, but nothing was taken.
The Muskegon Chronicle reports Thursday the reward amount likely will go up in the coming days if the crime isnt solved soon.
■ Related: Tips sought in shooting of jogger in western Michigan
"It makes no sense," said her uncle Tim Donkin. "Becky was not just a person running down the road she had a life. She did a lot to help people. And she just did a lot for everybody."
Police continue building a timeline of Bletsch's last hours. They say she contacted a family member Sunday afternoon around 5. One hour and ten minutes, later her body was found along Automobile road.
"Becky had a great love for her daughter Ellie. She put herself into everything she did," Donkin said.
Police continue to say Bletche's husband Kevin is not a suspect. They describe his alibi for Sunday night as "rock solid."
Donkin said he hopes police are closing in on a suspect, and he makes this plea on behalf of the Bletsch family. He asks that "if anybody has even an inkling to please contact the authorities. No matter how insignificant that it may be, if there's any inkling to please contact the Muskegon County Sheriff."
Those tips could bring answers to the questions Bletsch's family continue asking.
The Associated Press contributed to this report.


text:
Green Bay Packers tight end Jermichael Finley does a Lambeau Leap after scoring a touchdown Saturday during the Packers' Family Night scrimmage at Lambeau Field. Evan Siegle/Press-Gazette
If nothing else, the Green Bay Packers Family Night scrimmage provided a preview of how important Jermichael Finley has become to their offense.
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♦ Photos from Saturday night's scrimmage at Lambeau
♦ Photos of fans at Family Night
♦ Planning to see a practice? Check our schedule for dates and times
♦ Complete training camp coverage
♦ Sign up for Green Bay Packers text alerts.
The Packers third-year tight end was the best playmaker on the field against both the Packers No. 2 defense in normal scrimmage situations and the defensive starters in a two-minute drill. He finished with four receptions for a 13.8-yard average and one touchdown in relatively limited playing time, and was by far the standout player on the night.
You can throw the ball up to him and he just knows how to get it, safety Charlie Peprah said. A matchup nightmare. So Im glad hes on our team. Theres not too many people like him. Hes big enough where its hard for safeties to cover him, but hes still quick enough where hes a mismatch for linebackers.
If he keeps on developing and being a professional hell be a superstar in this league.
The Packers starting offense was on the field for only three series twice it started with the ball on its own 20 against the No. 2 defense, and then later a two-minute drill from its own 35 against the starting defense.
On the second series, Finley had two receptions on which his 6-foot-5 and 247-pound frame overwhelmed the 5-10, 198-pound safety Derrick Martin. The first was a 14-yard reception that converted a third down, the second was a 14-yard touchdown pass.
Later in the two-minute, he had an 11-yard reception on which he ran over cornerback Brandon Underwood to get out of bounds and stop the clock, and a 16-yarder against Bishop on the next snap to move the ball to the defenses 33. If this night was any indication, Finley could become a go-to receiver on third downs because he provides quarterback Aaron Rodgers with a huge, athletic target who can get to balls no one else on the field can.
Obviously he has a big body, cornerback Tramon Williams said. From what I saw the cornerbacks and safeties had great coverage on him, but hes got a big body, athletic guy. What can you do? Weve got a great quarterback putting the ball in the right spots. Its tough against him.
And with that receiving corps I just want to wait for the season. You dont want to get too excited but I see a lot there.
Though Finley had a big night, the Packers No. 1 offense produced only the one touchdown in three tries. Finleys touchdown came on the second of its two series against the No. 2 defense, and when it faced the starting defense in two-minute, it moved into scoring position but couldnt put the ball in the end zone after having a second-and-1 at the 5.
That marked the fourth time in training camp that the starters have faced off in two-minute, with the offense needing a touchdown, and the defense has won all four. But Williams said the two-minute is skewed in the defenses favor in practice.
If you go back and do the statistics from last year I think we stopped em a lot also, Williams said, they only won, you probably can count it on one hand. Its one of those things, we play against them every day so we know our offense and we know the receivers, so its tough on them. Its one of those deals you know the receivers, some sets you see you know what theyre going to do. Its repetition we see every day.
Rodgers finished the night 9 of 14 passing for 127 yards and a touchdown, which is a passer rating of 117.3 points.
The two-minute was a major emphasis going into last season, we definitely answered that bell, coach Mike McCarthy said. Id definitely rank Aaron one of the better two-minute quarterbacks Ive coached, even though its early in his career. Ive had an opportunity to work with him on a daily basis, Im very confident in him handling two-minute situations.
The Packers starting defense only played three series also and wasnt challenged by quarterback Matt Flynn and the No. 2 offense. Both of Flynns regular series against the starters ended in three-and-outs, with cornerback Brandon Underwood ending the second with an third-down interception that he returned for about a 30-yard touchdown.
In the two-minute against the starting offense, linebacker A.J. Hawk blew up a third-and-one run by halfback Ryan Grant for a four-yard loss, to the 9. Rodgers fourth-down pass to Charles Dillon, an undrafted rookie who is a long-shot to make the team, was incomplete.
The No. 1 defense played extremely well, McCarthy said. Go out and create three and outs, thats exactly what youre looking for.


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Satan is the father of lies: "You are of your father the devil, and you want to do the desires of your father. He was a murderer from the beginning and does not stand in the truth, because there is no truth in him. Whenever he speaks a lie, he speaks from his own nature; for he is a liar, and the father of lies" (John 8:44). Just as God's nature is truth (Titus 1:2), so Satan's nature is falsehood. It was by false witness that Satan tempted man into ruin.
Now the serpent was more cunning than any beast of the field which the Lord God had made. And he said to the woman, "Has God indeed said, 'You shall not eat of every tree of the garden'?" 2 And the woman said to the serpent, "We may eat the fruit of the trees of the garden; 3 but of the fruit of the tree which is in the midst of the garden, God has said, 'You shall not eat it, nor shall you touch it, lest you die.' " 4 Then the serpent said to the woman, "You will not surely die. 5 For God knows that in the day you eat of it your eyes will be opened, and you will be like God, knowing good and evil." 6 So when the woman saw that the tree was good for food, that it was pleasant to the eyes, and a tree desirable to make one wise, she took of its fruit and ate. She also gave to her husband with her, and he ate. ―Genesis 3:1-6
At the root of every sin is a lie. "For out of the heart come forth evil thoughts...false witness, railings: these are the things which defile the man" (Matt. 15:19-20).
The truth is simple; the lie is complicated. We don't trip when telling the truth but managing a lie can get out of control. The truth is easy to remember; the lie involves too many fabricated details. Thus, the false witness frequently stumbles and is revealed. "He who utters truth shows forth righteousness, but a false witness deceit.... The lip of truth shall be established forever, but a lying tongue is but for a moment" (Proverbs 12:17, 19).
God's word is always consistent with God's actions. We expect to see God act in a consistent way with His words. Since God's words and actions are consistent with one another, we say that God is trustworthy. Likewise, a man's words are indicative of his trustworthiness. You are as good as your word. A man who would lie would probably steal. In other words, a false witness cannot be trusted in any area of life. The so-called "little white lies" that we are prone to tell are also a reflection of our character. If you cannot be trusted in the little things, then why should you be trusted in the big things? The righteous man is described in Psalm 15 as one who "swears to his own hurt, and does not change."
Deception is lying to others and self-deception is lying to yourself; convincing yourself of a falsehood. Selfishness motivates sinners to believe what they want to believe about themselves. This is often done to justify some desire, attitude or action, and so, we spin the truth (which is a lie), in order to excuse ourselves. We engage in a cover-up. Truth loves the light, the lie loves the darkness.
Truthful words are essential to healthy relationships. "You shall not bear false witness against your neighbor" (Ex. 20:16; Dt. 5:20; Matt. 19:18). The tongue is our interface with the world. "For the mouth speaks out of that which fills the heart" (Matt. 12:34). We are called to "speak the truth in love" to one another (Eph. 4:15). "You shall not...lie one to another" (Leviticus 19:11). The power and character of your words either builds or destroys, "A man who bears false witness against his neighbor is a maul, and a sword, and a sharp arrow" (Proverbs 25:18). "You shall not take up a false report; put not your hand with the wicked to be an unrighteous witness" (Exodus 23:1). "Wherefore, putting away falsehood, speak the truth each one with his neighbor, for we are members one of another" (Ephesians 4:25). "Lie not one to another, seeing that you have put off the old man with his doings" (Colossians 3:9).
"There are six things which Jehovah hates, yes seven which are an abomination to Him: haughty eyes, a lying tongue...a false witness who utters lies" (Proverbs 6:16-19). Be scrupulous in what you say. Your word is one of the precious possessions you have. Treasure it.


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Time Warner VP pokes holes in NSA spy program
It is impossible to judge the constitutionality of he National Security Agency's electronic surveillance program without facts about the program, according to Time Warner Senior Vice President David Kris.
Kris testified for a Senate Judiciary Committee hearing Tuesday on the NSA program, which the White House claims targeted Americans in contact with foreign terrorists.
At Security Pipeline.
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What causes loss of appetite and low grade fever? 4 possible conditions
See a list of possible causes in order from the most common to the least.
Addison's disease occurs when the adrenal cortex is damaged and the adrenal glands don't produce enough of the steroid hormones cortisol and aldosterone.
Systemic lupus erythematosus (SLE) is an autoimmune disease and the most common type of lupus. One of its common symptoms is a rash on the cheeks and nose called a "butterfly rash."
Pernicious anemia (PA) is an autoimmune disorder in which the body fails to make enough healthy red blood cells (RBCs), resulting in vitamin B-12 deficiency. Most people experience a burning or sore tongue.
Please consult a healthcare professional if you have health concerns.
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Someone approached me about renting space on my blog (in the sidebar) for the purpose of advertising. Anyone have any experience with this? If so what about a fee structure? And most of all, is it a good idea?
Hope to hear from you.
Joyce at Joyce's Choices at http://www.jssherr.blogspot.com
Views: 96
It's not a bad idea, especially if you're being approached by people who want to do it. You can still control what you'll allow. If it doesn't cost you anything, why not?
There are a number of ad management tools that you can use to automate it, the easiest for someone who doesn't want to spend a lot of time on it is probably adpeeps, who'll host an ad server for you for $3.99/mo., so if you can get at least $4 a month out of the people asking for a spot, why not do it? Adpeeps lets you set CPM and CPC pricing and allows advertisers to create their own accounts on your branded ad server and pay for spots, with or without your intervention.
LOL this is probably all gobbledegook to you... If I'm right and it is, it's probably not worth managing it yourself unless the payoff is much larger than just a few bucks a month - so I'd just say "no" to the person asking, then use the AdSense Gadget instead and let Google worry about it and send you a check every once in awhile.
Hi Joyce!
There are a lot of ways you can set banner ad pricing, but the easiest way is to set a flat per month fee for a certain size graphic. When I was a design blogger, this is what I did... my traffic was around 4,000 unique visits a month and I sold a few side bar graphic banners for $40 a month. There's no set standard for what you charge for a banner ad, but higher traffic sites of course, are able to charge more and lower traffic sites charge less. I've seen people go as low as $25 for a 125x125 square side bar banner.
If you receive good traffic, and you're serious about making income from advertising on your blog, you should look into joining an ad network. Blogher runs a reputable one, and at one point I was a member of Clever Girls (who also offer opportunities to write sponsored posts). Other ad networks bloggers I know use include: CafeMom (http://www.cafemom.com/), Federated Media (http://www.federatedmedia.net/), and Social Spark (http://www.socialspark.com/).
The most important thing is making sure whatever ads you show are reputable and of interest to your readers. I would never join an ad network that doesn't give me control over which advertisements show up on my blog. I also don't really care for the link ads (which link to words within your copy) because I think it's distracting and spammy looking.
Here are some links that may help you learn more:
Final note... if your blog is hosted on a free blogging service, make sure you check the terms of use before adding any advertising. Wordpress.com for example, doesn't allow outside ad networks, although they do offer their own ad network that you can sign up for. Selling advertisement on a service that doesn't allow it can get your blog shut down. If you're self hosted, however, you don't have to worry about it.
Hope that helps some.
No, DO NOT GET MONEY INVOLVED. When you advertise on someone elses website, its more about forming bonds and connections with fellow book bloggers. When you add money to the equation it ends up being about business.
On top of that sidebar ads normally don't get allot of views, its completely depends on the blog. I suggest a blog button swap. You add my blog button, and I'll add yours- that kind of thing. Utterly money free.
- Eddie of http://editorsarticle.blogspot.ca
Follow my blog! or participate in a blog button swap!
I've got a banner exchange program going, too.
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The races for the 23 seats on the Oneida County Board of Legislators are starting to take shape.
The races for the 23 seats on the Oneida County Board of Legislators are starting to take shape.
This year's contests will be the first with the new, smaller Legislature size. The board will shrink from 29 to 23 members on Jan. 1, 2014, meaning several sitting legislators must battle one another in consolidated districts.
Separate from the shrinking of the board, the political parties are putting forward a busy field of candidates.
In total, there are at least 10 races shaping up for the fall. That's based on the current slate of endorsed candidates from the Republican, Democratic, Conservative and Independence parties.
Petitions to run are due to the county Board of Legislators on July 11, so a complete roster will be available then.
When the redistricting map was complete, there were six districts in which sitting legislators would be pitted against one another.
By now, however, some of those legislators have decided not to run.
For example, in the new 10th District, two veteran legislators, Republican Majority Leader George Joseph and former Republican Majority Leader David Wood, would have gone head to head. Wood, however, has opted to retire after 34 years.
Wood said he and Joseph discussed the situation and he'd made a choice.
Wood said he loves working with horses and as a member of the board, he was legally prevented from being directly involved in racing.
"I said at the end of the day, I'd rather go with the horses," he said.
Joseph isn't off the hook, however. Former Democratic Minority Leader Michael Hennessy, who left the board several years ago, is running on the Conservative line.
The major parties
The two major parties, the Democrats and the Republicans, are backing their own sitting legislators for the most part.
An exception, however, is the Democrats' choice not to support Legislator Harmony Speciale of West Utica.
They are instead endorsing Lori Washburn, the longtime ward chairwoman from that neighborhood.
"It's one thing to win the seat," Washburn said. "It's a whole other ballgame to keep it."
She said she doesn't think first-termer Speciale has been effective, and it's time for a change.
Speciale said she had worked hard to fix problems in the Democratic caucus and Washburn's endorsement was politics at its worst.
"I feel like it's because I stood up and confronted the issues that were going on and that's why I didn't get the endorsement," she said. "I have lost faith in the political process."
Speciale said she will likely run for her seat on an independent line.
The smaller parties
Page 2 of 2 - In an area where the Democratic and Republican parties are closely matched, endorsements by the smaller parties can mean a lot.
In Oneida County, there are 47,295 registered Democrats and 51,444 registered Republicans, according to the most recent state Board of Elections figures.
The Conservative Party has 2,009 registered members and the Independence Party 7,752, the figures show. Countywide, there are 25,316 registered voters with no party affiliation. No figures for the 23 legislative districts were available.
The redistricting also might open up new possibilities for the smaller parties to field candidates of their own.
With sitting legislators having to build name recognition in new territories, an outside candidate may have a better chance, officials from those parties said.
Both the Conservative and Independence party cross-endorsed some sitting Republican or Democratic legislators, but rejected others.
Conservative Party Chairwoman Julie Miller said her party opposes the agreement between the Oneida Indian Nation, the state and Oneida and Madison counties, and had not endorsed legislators who voted in favor of it.
She said in an email that the agreement "blatantly forfeits the rights of residents" and "allows for unlimited commercial development that will directly compete with taxpaying businesses in the county."
The Independence Party also based its endorsements on how closely candidates agreed with the party platform, Independence Party Utica chairman Tim Julian said.
For example, first-term Democrat David Gordon won the nod for a New Hartford seat over former Republican Majority Leader Jim D'Onofrio, because of Gordon's support for term limits.
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Tim Winton's The Turning
Dirs: Marieka Walsh, Warwick Thornton, Jub Clerc, Robert Connolly, Anthony Lucas, Rhys Graham, Ashlee Page, Tony Ayres, Claire McCarthy, Stephen Page, Shaun Gladwell, Mia Wasikowska, Simon Stone, David Wenham, Jonathan auf der Heide, Justin Kurtzel, Yaron Lifschitz, Ian Meadows. Australia. 2013. 179mins
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Panasonic GX 1 external view finder
Started Nov 27, 2012 | Questions thread
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Veteran Member • Posts: 5,679 • Gear list
bigbob33 wrote:
I bought the Panasonic Lumix gx1... Is it worth the money for the Dmw-lvf 2 viewfinder? bob
Depends on your shooting style. I've been using the older LVF1 on my LX5. Main advantage is quickness and accuracy of framing. When I'm trying to shoot moving subjects, I can track the action much better with my eye to the viewfinder rather than waving the camera at arm's length in front of me. Handy for street shooting. I also use it for shooting landscapes when I want to compose with extreme precision and when bright sunlight makes it hard to see the display screen. The LVF1 is pretty grainy. I'm looking forward to getting the LVF2 for my new LX7.
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Gambling chips stacked around roulette wheel on gaming table
- Filed Under
The Western Regional Off-Track Betting Corporation has announced its support for the Orleans County Legislature's vote to formally oppose a plan by the Seneca Nation of Indians to build a Las Vegas-style casino in Henrietta.
In expressing its support, WROTB said the Orleans County Legislature is the first body to formally oppose the expansion of gambling in Monroe County.
Orleans County is one of 17 municipalities that own Western Regional OTB. ...


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- Filed Under
Power outages scheduled for areas in Santa Rita, Tamuning
Guam Power Authority crews will be working on power lines and other hardware. Power will be shut off for their safety. Power outages are scheduled for the following dates and areas:
• Feb. 26: Some of the customers living along ...


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Comments by Chair&Puckleberry
I love him :)
And I do believe him.
This cover is really creepy.
And Will kinda looks like Joseph Gordon-Levitt,sorta..
I couldn't pay attention to the music because the video was so distracting.She has a great voice but this video is so stupid!
I think Spencer and Emily had the best dresses.
Toby didn't kill Alison.That would be to obvious.
Hanna and Lucas definitely should get together!
I don't really like Aria and Ezra. I think Aria should get with Sean.He's at least her age.
I don't think this is the last of Alex.
The dress does look nice but it would look better without the sleeves.
This episode was really good! I was kinda expecting more Puckleberry though :/
I'm glad Rachel and Jesse are over.Now he can go back to Vocal Adrenaline and like Puck and Rachel get togeher!:)
If only I owned the show. haha
I'm glad Nate kicked Jenny out.
She needs to die in a ditch!lol
The flashback of Blair and Chuck made me very sad.
ehh;I kinda like William but he is up to something obviously haha.
Vanessa and Dan kinda bored me!
I like Britney's old songs.They should sing those!
Like stronger,lucky,sometimes,etc.
David/Lauren:Finally back together!But of course Morgan and her dad have to come and mess that up!This has to be my favorite couple! Jonah/Riley:They were kinda boring!I can't believe Jonah would want Riley back.Is he crazy?Ella is freaking hot! Riley/Drew:They are starting to grow on me.I think I'm going to like Drew's storyline with his heart and stuff. Amanda/Ella:GO ELLA!She kicked ass. CW better renew Melrose Place for a second season.The show has become one of my favorites.
What the HELL is she wearing!


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Has she hit puberty yet??
Holy Crap! How old is she? 14?? She looks so young! Not that I'm surprised…Ovi seems like the type to go for jailbait
I'm just wonder why people posting her very old pictures? She's prolly really 14 on those pix but now she's something like 21 :S
They are a cute couple.
Duckface on film, really?
She´s 23 and she´s pregnant.
she's 23 ,born sept `87
Valeria is stunning beauty. They are great couple.
I am extremely inspired along with your writing skills and also with the structure to your blog.
Is this a paid theme or did you customize it your
self? Anyway keep up the nice quality writing, it is rare to look a
nice blog like this one nowadays..


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The Corners, owned by Charles Ready Sr., is getting a historical marker from the APTA on Jan. 6, 2013.
- Filed Under
Rutherford County Chapter of the Association for the Preservation of Tennessee Antiquities will host a winter marking of a historic home from 2-4 p.m. Jan. 6.
At the event, the Rutherford County Chapter of the APTA will place a marker at the "The Corners," built in 1829 by Charles Ready Sr. Following the marking ceremony, a tour and reception will be hosted by current owner Karen Ford.
"Karen, who owns Goodness Gracious Restaurant and Catering (located in downtown Murfreesboro) is making this house a new venue for her to expand her catering business with a venue," says Denise Carlton with the ...


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Whiplash is an injury to the soft tissues of the neck. Whiplash injury strains the muscles and ligaments of the neck beyond their normal range of motion. There is often pain and stiffness in the neck for the first few days following a whiplash injury. The pain can also be felt in the surrounding muscle groups in the head, chest, shoulders, and arms.
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Originally posted by Jim Petersson: I chose to use name + location as a primary key. Of course this has to be mentioned in the choices file. I guess this is one of those cases were you need to make a design decision for yourself.
Originally posted by Joshua Fix: I just call my find(String[]) method on the data that was passed in to the create method. It in turn attempts to match every field of a record to the fields of the record that is trying to be created. If any matches are returned, then there is an entry with all the same values. In that case, I throw a DuplicateKeyException.
Originally posted by Ewan Livingstone: I don't see any way in which there could ever be any circumstance in which one cannot insert a record because the ID/key already exists. Am I missing something?
Again: why shouldn't hotel "Palace" in "Sometown" have two (or more) rooms in the offering on the same date, for the same price, and both non-smoking? It's a hotel, so they're expected to have many rooms available! I think the only valid key is the record number. There is no reason to say that two records are not allowed to have all fields equal.
Originally posted by rinke hoekstra: Supposing you are talking about urlybird: I wouldn't do that. What makes you think that each hotel just has exactly one room to offer? I didn't think that very likely. It is more likely that, when a hotel is a customer of urlybird, they may have several rooms in the offering, and two rooms may even have exactly the same field values. Why shouldn't a hotel have two rooms available on the same date, both non-smoking, for the same price?
Originally posted by Joshua Fix: I am working on B&S. For me, it seemed too unlikely that two businesses with the same name are in the same city that offer the same specialties and have the same number of employees and same rate.


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Solar PV Potential Opportunities in Ontario
API is showing people of Ontario how to reap the rewards of the most lucrative tariff program in the world, through our own Solar Across Ontario initiative!
On September 24, 2009, the government has announced its much anticipated feed–in tariff program, which pays long-term premium prices for clean electricity, positioning the province as a world leader and eliciting international praise.
In the announcement, the government guarantees to purchase electricity generated from a 10Kw solar system on your roof for $0.802/kwh for 20 years!
For a more detailed Solar PV Potential Opportunities in Ontario, please visit www.solarpvlease.com.
*These results are from Federal Government endorsed simulation software results.
Cumulative Cash Flows
You will see below a sample of cumulative cash flows of a 10kW system when installed in the York Region area. The data is produced by the Retscreen, the climate data location used in this example is Toronto, Buttonville Airport, solar module used has the efficiency of 15.1%.
Payback Years
In the following table, the years of payback is summarized.
Estimated Income (Pre Tax) $
The following table, displays the comparison of the estimated income each system will generated. The figures are before taxes.
You may download the System Installations Comparison.
What are you waiting for?
Be the first in your area to make money from Solar PV.


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Door to Door Shipping
Most of the orders will send in 24 hours after payment confirmed.
We will inform you if we need more time to prepare.
It usually takes 5 to 8 working days by EMS
Shipping by DHL /TNT /UPS take 3-5working days.
(except regular inspected by customs, severe weather, flood, earthquake etc.)
All items you purchased from Afra under 1 year warranty.
Customer Service
We offer 7/24 full time service, Please feel free to contact me if you have any problem on my product.
Your inquiry will be replied within 6 hours.
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If you want to exchange the items received, you must contact us within 15days of the receipt of your order. And you should pay the additional shipping fees incurred and the items returned should be kept in their original status
Custom Service
The order will be declared as gift or lower value to eliminate&minimize your custom's tax, and easy the custom proceeding.
HOWEVER - In certain countries, you have to pay taxes or duties on imported goods. Sometimes goods under a certain value, or in certain categories, do not incur taxes.
The rules are different in every country. If you find out information about the import tax situation in your country, and you believe there are ways to minimize the taxes you have to pay (or eliminate the taxes altogether), just tell us what you need by putting instructions (regarding labeling, packing, declarations, invoices, etc.) in the comments field during checkout. We are more than happy to follow your instructions.
Feedback will be left in a timely manner after feedback has been received. Should there be any problems with your purchase, please do not leave neutral or negative feedback before contacting us for resolution. Any customer who leaves a neutral or negative feedback without giving us the opportunity to resolve the issue, We strive to make every transaction a positive experience and will do everything possible to correct any problem. thank you very much!
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Do you know the right medication for your children?
Asthma in young children is one of the most common causes of hospital admission and visits to the doctor in this age group.
However it is not easy to diagnose asthma in children under 5 years old as there are many reasons for wheezing and coughing at that age.
Once diagnosis is decided, often treatment is with trials of different doses of medication. Parents often ask about whether changes to a child's bedding or diet is needed.
The aim of good asthma management is to ensure that children can lead a normal healthy life, while taking only as much medication as is needed to keep them well, and avoid asthma attacks .
Children can also spend periods of time away from home. It is important that the people around your child are aware if they have asthma, and know how to recognise symptoms and what to do in an emergency. Parents can provide friends and family with a copy of their child's asthma action plan and asthma first aid information to help them be prepared.
Children's services and schools play an important role in supporting children with asthma, and they can also access resources and training.
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- KNIA / KRLS - http://kniakrls.com -
The Write Place Sponsoring Book Contest
Posted By News On October 2, 2011 @ 6:23 am In Today's Local News | Comments Disabled
Aspiring writers will have a chance to have their manuscript published for free in a contest sponsored by a local book publisher. Hannah Crawford with the Write Place in Pella says they developed the idea originally for the Dream Big Grow Here contest and though they did not win that contest, they decided to go ahead with the plan. All genres will be accepted. Rules for entering the contest will be released October 10th, when submissions can be made.
Article printed from KNIA / KRLS: http://kniakrls.com
URL to article: http://kniakrls.com/2011/10/the-write-place-sponsoring-book-contest/
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