the document is about chromosomal analysis technique named array CGH technology, the complete procedure and the result interpretation of chromosomal variation

1. Array CGH
Department of MMG
12/2/2020

2. Array CGH December 2, 2020
department of MMG
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Table of contents
Table: 01
Sr. no. Contents
01 Introduction
02 Size of probes
03 How does array CGH work
04 When to use Array CGH
05 Procedure
06 Explanation
07 Reporting Array CGH results
08 Webliography
09 References
Table: 02
Sr. no. List of figures
01 bands in the diagram of chromosome 16
02 two strands of DNA are held together by double helix
03 DNA strand showing nucleotide base pairs
04 an overview of array chg.
05 analysis of the results of array chg.

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Array-CGH (Comparative genomic hybridization)
Introduction:
Looking at chromosomes (chromosome analysis) Chromosomes
cannot be seen with the naked eye but if they are stained and
magnified under a microscope it is possible to see that each one has a
distinctive pattern of light and dark bands that look like horizontal
stripes. You can see these bands in the diagram of chromosome 16
shown in Figure 1. They are numbered outwards starting from where
the short and long arms meet (the centromere). By looking at your
child’s chromosomes in this way, often referred to as karyotyping, it is
possible if the change is large enough to see if there is a chromosome
imbalance (loss or gain of chromosome material) or if the
chromosome is rearranged in any way. Clinical scientists who do the
analysis are very skilled at detecting very small and often very subtle
changes. However, as the amount of material gained (duplicated) or
lost (deleted) can often be extremely small and impossible to see on a
routine chromosome test even by the most skilled of scientists, your
child may have been told their chromosome analysis was normal. The
clinician may have indicated that an underlying genetic basis was still
likely. The newest test now available for looking at chromosomes is
called a microarray-based comparative genomic hybridization (also
called an array CGH) test. (1)
Array-CGH was developed in the late nineties to detect DNA copy
number changes at high resolution along the genome or locus of
interest. (2)
Comparative genomic hybridization (CGH) to metaphase
chromosome targets has significantly contributed to our understanding of the cancer cytogenetics
of more complex malignancies such as the solid tumors. This molecular cytogenetics-based
technique (hereafter referred to as “chromosome CGH”) is capable of defining genome-wide
DNA copy number imbalances in sample cells relative to a normal reference in a single
experiment. (3)
Chromosome abnormalities can be associated with developmental delay, autism spectrum
disorder, learning difficulties, dysmorphic features and other congenital abnormalities. Array
CGH can detect smaller genetic changes than is possible by conventional karyotyping, and can
provide accurate information on the size and possible consequences of the gains (duplications) or
losses (deletions) identified. Multiple studies have shown that array CGH, when applied to
appropriate patients, will detect up to three times more pathogenic chromosome imbalances than
karyotyping due to its greater precision and sensitivity. Consortiums in the USA and many EU
countries have adopted array CGH as the front-line test in this patient cohort. Array CGH is now
more frequently used for prenatal studies as an adjunct or replacement for conventional
cytogenetic studies, particularly where structural fetal abnormalities are seen at ultrasound scan
but also at a patient’s or doctor’s request. The technique may also be used as a follow up test to
Figure 1; bands in the diagram of
chromosome 16

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characterize anomalies detected by a previous study (e.g. an apparently balanced de novo
rearrangement or marker chromosome). (4)
In an attempt to overcome some of the aforementioned limitations associated with traditional
CGH, investigators have developed a newer method that combines the principles of CGH with
the use of microarrays. Instead of using metaphase chromosomes, this method—which is known
as array CGH, or simply a CGH—uses slides arrayed with small segments of DNA as the targets
for analysis. These microarrays are created by the deposit and immobilization of small amounts
of DNA (known as probes) on a solid support, such as a glass slide, in an ordered fashion. (5)
Size of probes:
Probes vary in size from oligonucleotides manufactured to represent areas of interest (25–85
base pairs) to genomic clones such as bacterial artificial chromosomes (80,000–200,000 base
pairs). Because probes are several orders of magnitude smaller than metaphase chromosomes,
the theoretical resolution of a CGH is proportionally higher than that of traditional CGH. The
level of resolution is determined by considering both probe size and the genomic distance
between DNA probes. For example, a microarray with probes selected from regions across the
genome that are 1 Mb apart will be unable to detect copy number changes of the intervening
sequence. (6)
How does array CGH work?
A microarray works by exploiting the ability of a DNA molecule (or
strand) to bind specifically to, or hybridize to, another DNA molecule
(strand).
The DNA in our cells is arranged as a double helix (see Figure 2) in
which the two strands of DNA are bound (‘paired’) together by bonds
between the base pairs (bps). A single-stranded DNA fragment is made
up of four different building blocks (called nucleotides), adenine (A),
thymine (T), guanine (G), and cytosine (C) which are linked end to end.
Adenine (A) will always pair with thymine (T) and guanine (G) will
always pair with cytosine (C) (see Figure 3).
When to use Array CGH?
In postnatal cases, patients should present with one or more of the
following:
• Mental retardation
• Autism/autism spectrum disorder
• Developmental delay
• Dysmorphic features
In prenatal cases, patients may present with:
• Abnormalities or increased nuchal translucency on ultrasound scan
which may be associated with a chromosome imbalance.
• Congenital malformations
Figure 2; two strands of DNA are
held together by double helix
Figure 3 DNA strand showing
nucleotide base pairs

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Approximately 10–20% of results identify extra or missing DNA which may or may not be
relevant to the clinical phenotype, and will require further family studies to assist with
interpretation (7)
.
Array CGH (aCGH) has a much higher resolution than G-banded chromosome analysis and most
cytogenetic departments are now using this approach either as an adjunct to G-banded
chromosome analysis, or as a first-line test for selected patient groups. This service has been
offered since May 2008 using a patient vs patient (phenotype mismatched) hybridization strategy
to minimize costs, an important consideration in a state-funded health service; first line testing
by aCGH for all constitutional referrals for genome imbalance has been in place since September
2008.
Genomic DNA extracted from peripheral blood or saliva, or DNA provided by external
laboratories, was processed. Briefly, samples were co-hybridized with other samples mismatched
for phenotype and matched for sex (thus halving consumable costs compared with patient vs
control). Agilent 4x44k oligonucleotide array platform AMADID 017457 was initially used,
replaced in 2010 by an 8x60k platform (AMADID 028469) which included additional probes in
regions of clinical interest, and in the pseudoautosomal regions. Analysis was performed using
Agilent algorithm ADM-2, threshold 6 and a 3-probe minimum aberration call; a further analysis
using ADM-1 was carried out to maximize detection of mosaicism. Imbalances of regions
represented in the Database of Genomic Variants in at least three non-BAC based studies were
classified benign, and recorded but not reported. All samples with other imbalances were re-
tested using G-banded karyotyping, QF-PCR, FISH, custom MLPA or a repeat array (8)
.
Procedure:(step by step)
1. Regardless of the type of probe, the basic methodology for a CGH analysis is consistent.
2. First, DNA is extracted from a test sample (e.g., blood, skin, fetal cells).
3. The test DNA is then labeled with a fluorescent dye of a specific color, while DNA from
a normal control (reference) sample is labeled with a dye of a different color.
4. The two genomic DNAs, test and reference, are then mixed together and applied to
a microarray.
5. Next, digital imaging systems are used to capture and quantify the relative fluorescence
intensities of the labeled DNA probes that have hybridized to each target.
6. The fluorescence ratio of the test and reference hybridization signals is determined at
different positions along the genome, and it provides information on the relative copy
number of sequences in the test genome as compared to the normal genome.
7. The recent sequencing of the human genome and development of high-throughput
methods of robotically arraying genetic material on a solid surface have enabled the
detection of submicroscopic chromosomal deletions and duplications at an unprecedented
level (9)
Explanation:
When two complementary sequences find each other they will lock together, or hybridize. The
microarray comprises thousands of short sequences of DNA (“probes”), arranged in a precise
grid on a glass slide called a chip. DNA from the patient is “digested” (chopped up into short
lengths or fragments), then these fragments are labelled with a colored fluorescent dye.

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Reference DNA, from a person, or pool of people, with no genetic abnormalities, is labelled with
a different colored fluorescent dye. The fluorescent dyes commonly used are red and green.
Reference and patient samples are mixed together and applied to the chip and hybridization takes
place – the fragments of DNA hybridize with their matching probes on the array. The chip is
then scanned in a machine called a microarray scanner which measures the amount of red and
green fluorescence on each probe. For example, the patient sample may be labelled with a red
fluorescent dye and the reference sample may be labelled with green. The microarray scanner
together with computer analytical software calculates the ratio of the red to green fluorescent
dyes to determine whether, for the piece of DNA represented by each probe, the patient sample
has the correct amount of DNA (shown as yellow), too much DNA (a duplication) which would
be shown by too much red, or too little DNA (a deletion) shown by too much green.(9)
Reporting Array CGH results
arr [hg19] 16p11.2(29,673,954-30,198,600) x1
arr -- The analysis was by array-CGH
hg19 -- Human Genome build 19. This is the reference DNA sequence that the base pair
numbers refer to. As more information about the human genome is found, new “builds” of the
genome are made and the base pair numbers may be adjusted.
Figure 4; an overview of array chg.

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16p11.2 --- A change was found in band 16p11.2
(29,673,954-30,198,600) -- The first base pair shown to be missing is number 29,673,954
counting from the left of the chromosome. The last base pair shown to be missing is 30,198,600
X1-- means there is one copy of these base pairs, not two – one on each chromosome 16 – as you
would normally expect. (10) (11)
Webliography:
https://www.tdlpathology.com/specialties/genetics/array-cgh-testing/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3632487/
info@rarechromo.org
www.rarechromo.org
https://www.nature.com/scitable/topicpage/microarray-based-comparative-genomic-hybridization-
acgh-45432/#
https://www.researchgate.net/publication/24312773
References:
1. (DeRisi et al., 1996; Schena et al., 1995; Shaffer et al., 2007). Rare Chromosome
Disorder Support Group, PO Box 2189, Caterham, Surrey CR3 5GN, UK
Tel/Fax: +44(0)1883 330766
2. Comparative Genomic Hybridization: DNA labeling, hybridization and detection
Richard Redon, Tomas Fitzgerald, and Nigel P. Carter.
3. From: B. Beheshti, P.C. Park, I. Braude, J.A. Squire. “Microarray CGH”. In: Y.-
S. Fan (Ed.), Molecular Cytogenetics: Protocols and Applications: Humana Press,
2002.
4. https://www.tdlpathology.com/specialties/genetics/array-cgh-testing/ (The doctors
laboratory)
5. (Schena et al., 1995) (Lucito et al., 2003)
Figure 5; analysis of the results of array chg.

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6. Pinkel D, Segraves R, Sudar D, Clark S, Poole I, Kowbel D, Collins C, Kuo WL,
Chen C, Zhai Y, et al. High-resolution analysis of DNA copy number variation
using comparative genomic hybridization to microarrays. Nat Genet. 1998;
20:207–211. [PubMed: 9771718]
7. Microarray CGH Ben Beheshti, Paul C. Park, Ilan Braude, Jeremy A. Squire
Ontario Cancer Institute, Princess Margaret Hospital, University Health Network,
and Departments of Laboratory Medicine and Pathobiology, and Medical
Biophysics, Faculty of Medicine, University of Toronto, Ontario, Canada.
8. Array CGH as a first line diagnostic test in place of karyotyping for
postnatal referrals - results from four years’ clinical application for over
8,700 patients Joo Wook Ahn, 1
Susan Bint,2
Anne Bergbaum,2
Kathy
Mann,2
Richard P Hall,2
and Caroline Mackie Ogilvie1
9. Microarray-based Comparative Genomic Hybridization (aCGH) By: Aaron
Theisen, Ph.D. © 2008 Nature Education
Citation: Theisen, A. (2008) Microarray-based Comparative Genomic
Hybridization (aCGH). Nature
10. Kannan Thirumulu Ponnuraj (2011). Cytogenetic Techniques in Diagnosing
Genetic Disorders, Advances in the Study of Genetic Disorders, Dr. Kenji Ikehara
(Ed.), ISBN: 978-953-307-305-7, InTech,
11. Chial, H. (2008) Cytogenetic methods and disease: Flow cytometry, CGH, and
FISH. Nature Education 1(1):76