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Autor     Filomena Tiziana Papa
Titel    Interpretation of molecular imbalances detected by array-CGH analysis
Ort    Siena
Jahr    2010
Anmerkung    University of Siena Ph.D in Medical Genetics
URL    http://www3.unisi.it/ricerca/dottorationweb/genetica_medica/Tesi/Papa%20PhD%20thesis.pdf
Webcite    https://web.archive.org/web/20131126083540/http://www3.unisi.it/ricerca/dottorationweb/genetica_medica/Tesi/Papa%20PhD%20thesis.pdf

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In the 1990s the introduction of molecular cytogenetic techniques into the clinical laboratory setting represented a major advance in the ability to detect known syndromes and identify chromosomal rearrangements of unknown origin. Fluorescent in situ hybridization (FISH), which is the annealing of fluorescently labelled locus-specific probes to their complementary sequences in the genome, allowed for the detection of specific microdeletion syndromes (Trask 1991) (Fig.1b1-b2).

78. Trask BJ: Fluorescence in situ hybridization: applications in cytogenetics and gene mapping. Trends Genet 1991; 7: 149-154.

[Page 9]

In the 1990s the introduction of molecular cytogenetic techniques into the clinical laboratory setting represented a major advance in the ability to detect known syndromes and identify chromosomal rearrangements of unknown

[Page 10]

origin. FISH, which is the annealing of fluorescently labelled locus-specific probes to their complimentary [sic] sequences in the genome, allowed the detection of specific microdeletion syndromes. [3]


3. Trask, B.J., Fluorescence in situ hybridization: applications in cytogenetics and gene mapping. Trends Genet, 1991. 7(5): p. 149-54.

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Therefore this method is still predominantly used when the clinical phenotype is suggestive of a particular disorder. Several other FISH-based methods, including spectral karyotyping (SKY), multicolour FISH (m-FISH), and comparative genomic hybridization (CGH) have proven extremely useful in the identification of unknown chromosomal material. There are currently a number of commercially available FISH probes for the most common disorders and this method is still predominantly used when the clinical phenotype is suggestive of a particular disorder. Several other FISH-based methods, including spectral karyotyping (SKY), multicolour FISH (m-FISH), and comparative genomic hybridization (CGH) have proven extremely useful in the identification of unknown chromosomal material.
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SKY and m-FISH rely mainly on the principal of differentially labelling each chromosome using a unique combination of fluorochromes and are especially beneficial for identifying the origin and content of supernumerary marker chromosomes (SMCs) and complex chromosome rearrangements (CCRs) that involve more than two chromosomes (Fig.1c). SKY and m-FISH rely mainly on the principal of differentially labelling each chromosome using a unique combination of fluorochromes and are especially beneficial for identifying the origin and content of supernumerary marker chromosomes (SMCs) and complex chromosome rearrangements (CCRs) that involve more than two chromosomes.
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This method is very useful for determining the origin of unknown genetic material, such as SMCs and other unbalanced rearrangements. However, CGH does not detect balanced rearrangements, the resolution is on the order of 5–10 Mb and consequently many genomic disorders cannot be detected (Yunis 1976). The need to screen the whole genome at a resolution that surpassed the existing technologies led to the implementation of microarray based CGH. The principle is very similar to that employed for traditional CGH, where two differentially labelled specimens are co-hybridized in the presence of Cot1 DNA (Fig.2).

88. Yunis J: High resolution of human chromosomes. Science 1976; 191: 1268-1270.

[Page 10]

This method allows the investigation of the whole genome and is very useful for determining the origin of unknown genetic material, such as SMCs and other unbalanced rearrangements. However, CGH does not detect balanced rearrangements, the resolution is on the order of 5–10 Mb and consequently many genomic disorders cannot be detected. [4]

[Page 12]

The need to screen the whole genome at a resolution that surpassed the existing technologies led to the implementation of microarray based CGH. The principle is very similar to that employed for traditional CGH, where two differentially labelled specimens are co-hybridized in the presence of Cot1 DNA.


4. Edelmann, L. and K. Hirschhorn, Clinical utility of array CGH for the detection of chromosomal imbalances associated with mental retardation and multiple congenital anomalies. Ann N Y Acad Sci, 2009. 1151: p. 157-66.

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Fig.2 Schematic representation of an array-CGH experiment. Test and reference DNA are differentially labelled, co-precipitated and hybridised to an array. After wash procedures, the slides are analysed through a scanner and fluorescence intensities of each probe are determined. After imaging processing and data normalization, the log2 ratios of the probes are plotted as a function of chromosomal position. Probes with a value of zero represent equal fluorescence intensity ratio between sample and reference. In this representation, copy number loss shift the ratio to the left and copy number gains shift the ratio to the right. Fig. 2 Schematic representation of an array-CGH experiment. a) Test and reference DNA are differentially labelled, co-precipitated and hybridised to an array. b) and c) After wash procedures, the slides are analysed through a scanner and fluorescence intensities of each probe are determined. d) After imaging processing and data normalization, the log2 ratios of the probes are plotted as a function of chromosomal position. Probes with a value of zero represent equal fluorescence intensity ratio between sample and reference. [...] In this representation, copy number loss shift the ratio to the left and copy number gains shift the ratio to the right.
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1.2 Array – CGH Methodologies

[...]

Two major types of array targets are currently being utilized. Initially, bacterial artificial chromosomes (BACs) were the array target of choice (Pinkel 1998). However, now oligonucleotide arrays have been adopted due to the increased genome coverage they afford. The design of both array types was made possible by the availability of the complete map and sequence of the human genome. The BAC arrays may contain DNA isolated from large insert clones that range in size from 150–200 kb, spotted directly onto the array or may employ the spotting of PCR products amplified from the BAC clones (Ylstra 2006). These arrays are generally very sensitive and results can be directly validated with FISH using the BAC DNA as a probe. However, production of BAC DNA is labor-intensive, and the resolution is limited to 50–100 kb, even on a whole genome tiling path array (Ishkanian 2004). Oligonucleotide arrays offer a flexible format with the potential for very high [resolution and customization.]


30. Ishkanian A.S. et al. 2004. A tiling resolution DNA microarray with complete coverage of the human genome. Nat. Genet. 36: 299–303.

60. Pinkel D. et al. 1998. High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays. Nat. Genet. 20: 207–211.

87. Ylstra B. et al. 2006. BAC to the future! Or oligonucleotides: A perspective for micro array comparative genomic hybridization (array CGH). Nucleic Acids Res. 34: 445–450.

1.2 Array – CGH Methodologies

Two major types of array targets are currently being utilized. Initially, bacterial artificial chromosomes (BACs) were the array target of choice. [6] However, more recently, oligonucleotide arrays have been adopted due to the increased genome coverage they afford. The design of both array types was made possible by the availability of the complete map and sequence of the human genome. The BAC arrays may contain DNA isolated from large insert clones that range in size from 150–200 Kb, spotted directly onto the array or may employ the spotting of PCR products amplified from the BAC clones. [8] These arrays are generally very sensitive and results can be directly validated with FISH using the BAC DNA as a probe. However, production of BAC DNA is labor-intensive and the resolution is limited to 50–100 Kb, even on a whole genome tiling path array. [9] Oligonucleotide arrays offer a flexible format with the potential for very high resolution and customization.


6. Pinkel, D., et al., High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays. Nat Genet, 1998. 20(2): p. 207-11.

8. Ylstra, B., et al., BAC to the future! or oligonucleotides: a perspective for micro array comparative genomic hybridization (array CGH). Nucleic Acids Res, 2006. 34(2): p. 445-50.

9. Ishkanian, A.S., et al., A tiling resolution DNA microarray with complete coverage of the human genome. Nat Genet, 2004. 36(3): p. 299-303.

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Several different platforms are available for oligonucleotide arrays that range from 25- to 85mers in length, some of which were adapted from genome-wide SNP-based oligonucleotide markers and others that were created from a library of virtual probes that span the genome, and consequently can be constructed to have extremely high resolution (Shaikh 2007). Both BAC and oligonucleotide arrays have been used successfully to detect copy number changes in patients with intellectual deficit (ID), multiple congenital anomalies (MCA) and autism. A number of different array design approaches have been taken for diagnostic purposes. A targeted array is one that contains specific regions of the genome, such as the sub-telomeres and those responsible for known microdeletion/microduplication syndromes, but does not have probes that span the whole genome (Bejjani 2005, Bejjani 2006, Shaffer 2006). A whole genome or tiling path array offers full genome coverage with different resolution.

8. Bejjani, B.A. & L.G. Shaffer. 2006. Application of array-based comparative genomic hybridization to clinical diagnostics. J. Mol. Diagn. 8: 528–533.

9. Bejjani, B.A. et al. 2005. Use of targeted array-based CGH for the clinical diagnosis of chromosomal imbalance: Is less more? Am. J. Med. Genet. A 134: 259–267.

70. Shaffer L.G. et al. 2006. Targeted genomic microarray analysis for identification of chromosome abnormalities in 1500 consecutive clinical cases. J. Pediatr.149: 98–102.

72. Shaikh, T.H. 2007. Oligonucleotide arrays for highresolution analysis of copy number alteration in mental retardation/multiple congenital anomalies. Genet. Med. 9: 617–625.

[Page 13]

Several different platforms are available for oligonucleotide arrays, some of which were adapted from genome wide SNP-based oligonucleotide markers and others that were created from a library of virtual probes that span the genome, and consequently can be constructed to have extremely high resolution. [10] Both BAC and

[Page 14]

oligonucleotide arrays have been used successfully to detect copy number changes in patients with ID/MCA and autism. A number of different array design approaches have been taken for diagnostic purposes. A targeted array is one that contains specific regions of the genome, such as the subtelomeres and those responsible for known microdeletion/microduplication syndromes, but does not have probes that span the whole genome. [11] [12] [13] [...] A whole genome or tiling path array offers full genome coverage with a resolution that is dependent on the spacing of the probes.


10. Shaikh, T.H., Oligonucleotide arrays for high-resolution analysis of copy number alteration in mental retardation/multiple congenital anomalies. Genet Med, 2007. 9(9): p. 617-25.

11. Bejjani, B.A., et al., Use of targeted array-based CGH for the clinical diagnosis of chromosomal imbalance: is less more? Am J Med Genet A, 2005. 134(3): p. 259-67.

12. Bejjani, B.A. and L.G. Shaffer, Application of array-based comparative genomic hybridization to clinical diagnostics. J Mol Diagn, 2006. 8(5): p. 528-33.

13. Shaffer, L.G., Risk estimates for uniparental disomy following prenatal detection of a nonhomologous Robertsonian translocation. Prenat Diagn, 2006. 26(4): p. 303-7.

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Therefore, 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 aetiology, to a "genotype-first" approach, in which a collection of individuals with similar copy-number imbalances can be examined for common clinical features (Neill 2010).

56. Neill N.J., et al., Comparative analysis of copy number detection by whole-genome BAC and oligonucleotide array CGH. Mol Cytogenet, 2010. 3: p. 11.

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. [38]

38. Neill, N.J., et al., Comparative analysis of copy number detection by whole-genome BAC and oligonucleotide array CGH. Mol Cytogenet, 2010. 3: p. 11.

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The text can also be found in the source given: see Mmu/Fragment_010_10b

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A more complete understanding of the full clinical spectrum of these disorders will be achieved as the use of aCGH in the clinic becomes more prevalent and as correlations of these clinical findings with the genomic lesions are made. A more complete understanding of the full clinical spectrum of these disorders will be achieved as the use of aCGH in the clinic becomes more prevalent and as correlations of these clinical findings with the genomic lesions are made.
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The clinical phenotypes associated with the reciprocal microduplications of the same genomic regions are, however, less well characterized probably because, in general, individuals with duplications tend to have a milder phenotype than those with the complementary deletions and this milder phenotype may not lead to clinical investigation (Van der Aa 2009; Hassed 2004; Potocki 2000). The introduction of aCGH in clinical practice has showed that the frequency of these duplications is much higher than heretofore appreciated. As aCGH becomes the primary method of testing individuals with even mild intellectual deficit/developmental delay (ID/DD), the frequency of microduplications at the common microdeletion syndrome loci will likely increase (Bejjani and Shaffer 2008).

7. Bejjani, B.A. & L.G. Shaffer, Clinical utility of contemporary molecular cytogenetics. Annu Rev Genomics Hum Genet, 2008. 9: p. 71-86.

27. Hassed S.J., et al., A new genomic duplication syndrome complementary to the velocardiofacial (22q11 deletion) syndrome. Clin Genet, 2004. 65(5): p. 400-4.

61. Potocki L, et al. Molecular mechanism for duplication 17p11.2-the homologous recombination reciprocal of the Smith-Magenis microdeletion. Nat Genet 2000;24:84–7.

81. Van der Aa N., et al., Fourteen new cases contribute to the characterization of the 7q11.23 microduplication syndrome. Eur J Med Genet, 2009. 52(2-3): p. 94-100.

However, duplications have not been observed until fairly recently, likely because, in

general, individuals with duplications tend to have a milder phenotype than those with the complementary deletions [43] [44] [45] [46] [47] and this milder phenotype may not lead to clinical investigation. [48] [49] The introduction of aCGH in clinical practice has virtually eliminated all the technical impediments of traditional cytogenetics and FISH and allowed the detection of such conditions with relative-but not complete-independence from the clinician's diagnostic judgment. Therefore, recent reviews of cohorts of patients ascertained with aCGH showed that the frequency of these duplications is much higher than heretofore appreciated. As aCGH becomes the primary method of testing individuals with even mild DD/ID, the frequency of microduplications at the common microdeletion syndrome loci will likely increase. [37] [50]


37. Bejjani, B.A. and L.G. Shaffer, Clinical utility of contemporary molecular cytogenetics. Annu Rev Genomics Hum Genet, 2008. 9: p. 71-86.

43. Berg, J.S., et al., Speech delay and autism spectrum behaviors are frequently associated with duplication of the 7q11.23 Williams-Beuren syndrome region. Genet Med, 2007. 9(7): p. 427-41.

44. Potocki, L., et al., Characterization of Potocki-Lupski syndrome (dup(17)(p11.2p11.2)) and delineation of a dosage-sensitive critical interval that can convey an autism phenotype. Am J Hum Genet, 2007. 80(4): p. 633-49.

45. Yobb, T.M., et al., Microduplication and triplication of 22q11.2: a highly variable syndrome. Am J Hum Genet, 2005. 76(5): p. 865-76.

46. Brunetti-Pierri, N., et al., Recurrent reciprocal 1q21.1 deletions and duplications associated with microcephaly or macrocephaly and developmental and behavioral abnormalities. Nat Genet, 2008. 40(12): p. 1466-71.

47. Van der Aa, N., et al., Fourteen new cases contribute to the characterization of the 7q11.23 microduplication syndrome. Eur J Med Genet, 2009. 52(2-3): p. 94-100.

48. Hassed, S.J., et al., A new genomic duplication syndrome complementary to the velocardiofacial (22q11 deletion) syndrome. Clin Genet, 2004. 65(5): p. 400-4.

49. Potocki, L., et al., Molecular mechanism for duplication 17p11.2- the homologous recombination reciprocal of the Smith-Magenis microdeletion. Nat Genet, 2000. 24(1): p. 84-7.

50. de Vries, B.B., et al., Diagnostic genome profiling in mental retardation. Am J Hum Genet, 2005. 77(4): p. 606-16.

Anmerkungen

Shortened but otherwise nearly identical. The list of references has been shortened, too, but here also copying has taken place (detectable by the placement of the year - which this time is not right behind the authors' names - and the usage of "p." in front of the pagenumbers, which is not done by Mmu in general). Nothing has been marked as a citation.

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3) MATERIALS & METHODS

3.1 Patients collection

Patients with ID and MCA enrolled in this study have been selected among those referred the Medical Genetics Unit of the University Hospital of Siena. All patients were evaluated by clinical geneticists.

3.2 Array-based CGH

3.2.1 Samples preparation

Genomic DNA of normal controls was obtained from Promega. Genomic DNAs were extracted from peripheral blood samples using a QIAamp DNA Blood Maxi kit according to the manufacturer protocol (Qiagen, www.qiagen.com). The OD260/280 method on a photometer was employed to determine the appropriate DNA concentration (Sambrook 1989). Patient and control DNA samples were sonicated to produce a homogeneous smear DNA extending from approximately 600 bp to 2 Kb. DNA samples were then purified using the DNA Clean and Concentrator kit (Zymo Research, Orange, CA). Ten micrograms of genomic DNA both from the patient and from the control were sonicated. Test and reference DNA samples were subsequently purify using dedicated columns (DNA Clean and Concentrator, Zymo research, CA92867-4619, USA) and the appropriate DNA concentrations were determine by a DyNA Quant™ 200 Fluorometer (GE Healthcare).

3.2.2 Human oligonucleotides array

Array based CGH analysis was performed using commercially available oligonucleotide microarrays containing about 43,000 60-mer probes with an estimated average resolution of about 100-130 Kb (Human Genome CGH Microarray 44B Kit, Agilent Technologies) and microarrays containing 99,000 60- mer probes with an estimate average resolution of 50-65 Kb (Human Genome CGH [Microarray 105A Kit, Agilent Technologies).]


67. Sambrook, J. & M.J. Gething, Protein structure. Chaperones, paperones. Nature, 1989. 342(6247): p. 224-5.

3. MATERIALS & METHODS


3.1 Patients collection Patients with ID and MCA enrolled in this study have been selected among those attending the Medical Genetics Unit of the University Hospital of Siena. All they were evaluate in genetic counseling and a clinically recognizable condition was excluded a diagnosis of a recognizable syndrome, and all patients.

3.2 Array-based CGH

3.2.1 Samples preparation

Genomic DNA of normal controls was obtained from Promega. Genomic DNA was extracted from peripheral blood using a QIAamp DNA Blood Maxi kit according to the manufacturer protocol (Qiagen, www.qiagen.com). The OD260/280 method on a photometer was employed to determine the appropriate DNA concentration. [94] Patient and control DNA samples were sonicated to produce a homogeneous smear DNA extending from approximately 600 bp to 2 Kb. DNA samples were then purified using the DNA Clean and Concentrator kit (Zymo Research, Orange, CA). Ten micrograms of genomic DNA both from the patient and from the control were sonicated. Test and reference DNA samples were subsequently purify using dedicated columns (DNA Clean and Concentrator, Zymo research, CA92867-4619, USA) and the appropriate DNA concentrations were determine by a DyNA Quant™ 200 Fluorometer (GE Healthcare).

3.2.2 Human oligonucleotides array

Array based CGH analysis was performed using commercially available oligonucleotide microarrays containing about 43,000 60-mer probes with an

[page 29:]


estimated average resolution of about 100-130 Kb (Human Genome CGH Microarray 44B Kit, Agilent Technologies) and microarrays containing 99,000 60-mer probes with an estimate average resolution of 50-65 Kb (Human Genome CGH Microarray 105A Kit, Agilent Technologies).


94. Sambrook, J. and M.J. Gething, Protein structure. Chaperones, paperones. Nature, 1989. 342(6247): p. 224-5.

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Physical positions of the probes correspond to the UCSC genome browser - GRCh build 37, Feb 2009 (http://genome.ucsc.edu). DNA labelling was executed essentially according to the Agilent protocol (Oligonucleotide Array-Based CGH for Genomic DNA Analysis 2.0v) using the Bioprime DNA labelling system (Invitrogen). Genomic DNA (2 μg) was mixed with 20 μl of 2.5X Random primer solution (Invitrogen) and MilliQ water to a total volume of 41 μl. The mix was denaturated at 95° C for 7 minutes and then incubated in ice/water for 5 minutes. Each sample was added with 5 μl of 10X dUTP nucleotide mix (1.2 mM dATP, dGTP, dCTP, 0.6 mM dTTP in 10 mM Tris pH 8 and 1 mM EDTA), 2.5 μl of Cy5-dUTP (test sample) or 2.5 μl of Cy3-dUTP (reference sample) and with 1.5 μl of Exo-Klenow (40 U/μl, Invitrogen). Labeled samples were subsequently purified using CyScribe GFX Purification kit (Amersham Biosciences) according to the manufacturer protocol. Test and reference DNA were pooled and mixed with 50 μg of Human Cot I DNA (Invitrogen), 50 μl of Blocking buffer (Agilent Technologies) and 250 μl of Hybridization buffer (Agilent Technologies). Before hybridization to the array the mix was denatured at 95°C for 7 minutes and then pre-associated at 37°C for 30 minutes. Probes were applied to the slide using an Agilent microarray hybridization station. Hybridization was carried out for 24/40 hrs at 65°C in a rotating oven (20 rpm). The array was disassembled and washed according to the manufacturer protocol with wash buffers supplied with the Agilent kit. The slides were dried and scanned using an Agilent G2565BA DNA microarray scanner. Image analysis was performed using the CGH Analytics software v.3.4.40 with default settings. The software automatically determines the fluorescence intensities of the spots for both fluorochromes performing background subtraction and data normalization, and compiles the data into a spreadsheet that links the fluorescent signal of every oligo on the array to the oligo name, its position on the array and its position in the genome. The linear order of the oligos is reconstituted in the ratio plots consistent with an ideogram. The ratio plot is arbitrarily assigned such that gains and losses in DNA copy number at a particular locus are observed as a deviation of the ratio plot from a modal value of 1.0. Physical positions of the probes correspond to the UCSC genome browser - NCBI build 36, March 2006. (http://genome.ucsc.edu). DNA labelling was executed essentially according to the Agilent protocol (Oligonucleotide Array-Based CGH for Genomic DNA Analysis 2.0v) using the Bioprime DNA labelling system (Invitrogen). Genomic DNA (2 μg) was mixed with 20 μl of 2.5X Random primer solution (Invitrogen) and MilliQ water to a total volume of 41 μl. The mix was denaturated at 95° C for 7 minutes and then incubated in ice/water for 5 minutes. Each sample was added with 5 μl of 10X dUTP nucleotide mix (1.2 mM dATP, dGTP, dCTP, 0.6 mM dTTP in 10 mM Tris pH 8 and 1 mM EDTA), 2.5 μl of Cy5-dUTP (test sample) or 2.5 μl of Cy3-dUTP (reference sample) and with 1.5 μl of Exo-Klenow (40 U/μl, Invitrogen). Labeled samples were subsequently purified using CyScribe GFX Purification kit (Amersham Biosciences) according to the manufacturer protocol. Test and reference DNA were pooled and mixed with 50 μg of Human Cot I DNA (Invitrogen), 50 μl of Blocking buffer (Agilent Technologies) and 250 μl of Hybridization buffer (Agilent Technologies). Before hybridization to the array the mix was denatured at 95°C for 7 minutes and then pre-associated at 37°C for 30 minutes. Probes were applied to the slide using an Agilent microarray hybridization station. Hybridization was carried out for 24/40 hrs at 65°C in a rotating oven (20 rpm). The array was disassembled and washed according to the manufacturer protocol with wash buffers supplied with the Agilent kit. The slides were dried and scanned using an Agilent G2565BA DNA microarray scanner. Image analysis was performed using the CGH Analytics software v. 3.4.40 with default settings. The software automatically determines the fluorescence intensities of the spots for both fluorochromes performing

[page 30:]

background subtraction and data normalization, and compiles the data into a spreadsheet that links the fluorescent signal of every oligo on the array to the oligo name, its position on the array and its position in the genome. The linear order of the oligos is reconstituted in the ratio plots consistent with an ideogram. The ratio plot is arbitrarily assigned such that gains and losses in DNA copy number at a particular locus are observed as a deviation of the ratio plot from a modal value of 1.0.

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3.3 Real-time quantitative PCR

Some aCGH data were confirmed by Real-time Quantitative PCR experiments. To design adequate probes in different regions of the human genome, we used an TaqMan Gene Expression Assays by design which provides pre-designed primers-probe set for real-time PCR experiments (Applied Biosystems, https://products.appliedbiosystems.com). PCR was carried out using an ABI prism 7000 (Applied Biosystems) in a 96-well optical plate with a final reaction volume of 50 μl. A total of 100 ng (10 μl) was dispensed in each of the four sample wells for quadruplicate reactions. Thermal cycling conditions included a pre-run of 2 min at 50°C and 10 min at 95°C. Cycle conditions were 40 cycles at 95°C for 15 sec and 60°C for 1 min according to the TaqMan Universal PCR Protocol (ABI). The TaqMan Universal PCR Master Mix and Microamp reaction tubes were supplied by Applied Biosystems. The starting copy number of the unknown samples was determined using the comparative Ct method as previously described (Ariani 2004).

3.4 Multiplex Ligation-dependent Probe Amplification (MLPA)

MLPA analysis was performed according to the provider’s protocol with a specifically designed set of probes for testing critical regions in DiGeorge syndrome (SALSA P023 kit; MRC-Holland, Amsterdam, Netherlands; http://www.mrc-holland.com), 1p-deletion syndrome, Williams syndrome, Smith-Magenis syndrome, Miller-Dieker syndrome, DiGeorge syndrome, Prader-Willi syndrome, Alagille syndrome, Saethre-Chotzen syndrome, Sotos syndrome: (SALSA P064B MR1 kit) and subtelomere regions (SALSA P036D subtelomeric primer kit). The ligation products were amplified by PCR using the common primer set with the 6-FAM label distributed by the supplier. Briefly, 100 ng of genomic DNA was diluted with TE buffer to 5 μl, denatured at 98°C for 5 minutes and hybridized with SALSA Probe-mix at 60°C overnight. Ligase-65 mix was then added and ligation was performed at 54°C for 15 minutes. The ligase was successively inactivated by heat, 98°C for 5 minutes. PCR reaction was performed in a 50 μl volume. Primers, dNTP and polymerase were added and amplification was carried out for 35 cycles (30 seconds [at 95°C, 30 seconds at 60°C and 60 seconds at 72°C).]


2. Ariani, F., et al., Real-time quantitative PCR as a routine method for screening large rearrangements in Rett syndrome: Report of one case of MECP2 deletion and one case of MECP2 duplication. Hum Mutat, 2004. 24(2): p. 172-7.

3.3 Real-time quantitative PCR

Some aCGH data were confirmed by Real-time Quantitative PCR experiments. To design adequate probes in different regions of the human genome, we used an TaqMan Gene Expression Assays by design which provides pre-designed primers-probe set for real-time PCR experiments (Applied Biosystems, https://products.appliedbiosystems.com) PCR was carried out using an ABI prism 7000 (Applied Biosystems) in a 96-well optical plate with a final reaction volume of 50 μl. A total of 100 ng (10 μl) was dispensed in each of the four sample wells for quadruplicate reactions. Thermal cycling conditions included a prerun of 2 min at 50°C and 10 min at 95°C. Cycle conditions were 40 cycles at 95°C for 15 sec and 60°C for 1 min according to the TaqMan Universal PCR Protocol (ABI). The TaqMan Universal PCR Master Mix and Microamp reaction tubes were supplied by Applied Biosystems. The starting copy number of the unknown samples was determined using the comparative Ct method as previously described. [95]

[page 31:]

3.4 Multiplex Ligation-dependent Probe Amplification (MLPA)

MLPA analysis was performed according to the provider’s protocol with a specifically designed set of probes for testing critical regions in DiGeorge syndrome (SALSA P023 kit; MRC-Holland, Amsterdam, Netherlands; http://www.mrc-holland.com), 1p-deletion syndrome, Williams syndrome, Smith- Magenis syndrome, Miller-Dieker syndrome, DiGeorge syndrome, Prader-Willi syndrome, Alagille syndrome, Saethre-Chotzen syndrome, Sotos syndrome: (SALSA P064B MR1 kit) and subtelomere regions (SALSA P036D subtelomeric primer kit). The ligation products were amplified by PCR using the common primer set with the 6-FAM label distributed by the supplier. Briefly, 100 ng of genomic DNA was diluted with TE buffer to 5 μl, denatured at 98°C for 5 minutes and hybridized with SALSA Probe-mix at 60°C overnight. Ligase-65 mix was then added and ligation was performed at 54°C for 15 minutes. The ligase was successively inactivated by heat, 98°C for 5 minutes. PCR reaction was performed in a 50 μl volume. Primers, dNTP and polymerase were added and amplification was carried out for 35 cycles (30 seconds at 95°C, 30 seconds at 60°C and 60 seconds at 72°C).


95. Ariani, F., et al., Real-time quantitative PCR as a routine method for screening large rearrangements in Rett syndrome: Report of one case of MECP2 deletion and one case of MECP2 duplication. Hum Mutat, 2004. 24(2): p. 172-7.

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Amplification products were identified and quantified by capillary electrophoresis on an ABI 310 genetic analyzer, using GENESCAN software (version 3.7) all from Applied Biosystems (Foster City, CA, USA). The peak areas of the PCR products were determined by GENOTYPER software (Applied Biosystems). A spreadsheet was developed in MicrosoftTM Excel in order to process the sample data efficiently. Data were normalized by dividing each probe’s peak area by the average peak area of the sample. This normalized peak pattern was divided by the average normalized peak pattern of all the samples in the same experiment (Koolen 2004).

36. Koolen, D.A., et al., Screening for subtelomeric rearrangements in 210 patients with unexplained mental retardation using multiplex ligation dependent probe amplification (MLPA). J Med Genet, 2004. 41(12): p. 892-9.

Amplification products were identified and quantified by capillary electrophoresis on an ABI 310 genetic analyzer, using GENESCAN software (version 3.7) all from Applied Biosystems (Foster City, CA, USA). The peak areas of the PCR products were determined by GENOTYPER software (Applied Biosystems). A spreadsheet was developed in MicrosoftTM Excel in order to process the sample data efficiently. Data were normalized by dividing each probe’s peak area by the average peak area of the sample. This normalized peak pattern was divided by the average normalized peak pattern of all the samples in the same experiment. [96]

96. Koolen, D.A., et al., Screening for subtelomeric rearrangements in 210 patients with unexplained mental retardation using multiplex ligation dependent probe amplification (MLPA). J Med Genet, 2004. 41(12): p. 892-9.

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Array-CGH analysis was performed using commercially available oligonucleotide microarrays containing about 105.000 60-mer probes (Human Genome CGH Microarray 105K Kit respectively, Agilent Technologies, Santa Clara, California) as previously reported by Pescucci et al.

17. Pescucci C, Caselli R, Grosso S, et al. 2q24-q31 deletion: report of a case and review of the literature. Eur J Med Genet 2007;50(1):21-32.

Array-CGH analysis was performed using commercially available oligonucleotide microarrays containing about 43.000 60-mer probes (Human Genome CGH Microarray 44B Kit, Agilent Technologies, Santa Clara, California) as previously reported.[6]

6.Pescucci C, Caselli R, Grosso S, Mencarelli MA, Mari F, Farnetani MA, Piccini B, Artuso R, Bruttini M, Priolo M, Zuffardi O, Gimelli S, Balestri P, Renieri A. 2q24-q31 deletion: report of a case and review of the literature. Eur J Med Genet 2007;50:21-32.

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