Indian Journal of Experimental Biology Vol. 47, October 2009, pp. 779-791
Review Article
Comparative genomic hybridization array study and its utility in detection of constitutional and acquired anomalies Joris Andrieux & Frenny Sheth* Laboratory of Medical Genetics, Jeanne de Flandre Hospital CHRU de Lille, Lille Cedex, France The last decade has witnessed an upsurge in the knowledge of cytogenetic disorders and putting the old technology in a new basket with molecular genetics. As conventional cytogenetic can detect the genetic alteration of 10-15 Mb, many of the micro-deletions and micro-duplications are missed. However, with the advent of technology of fluorescence in situ hybridization (FISH), the resolution of genetic aberrations can reach to 3-5 Mb, nonetheless the anomalies smaller than the above, need further precision which has been achieved using comparative genomic hybridization array (CGH-array). Introduction of array-CGH has brought higher sensitivity with automated DNA fragment analyzer and DNA chip for submicroscopic chromosomal anomalies that are missed till date in many of the acquired and constitutional genetic disorders. The resolution of the technology varies from several Kb to 1 Mb depending upon the type of array selected. With the recent improvement in the array-CGH technology, a link between cytogenetic and molecular biology has been established without replacing conventional cytogenetic technique. The wider accessibility of the technology shall certainly provide a clue to the many unidentified/unexplained genetic disorders which shall prove to be a boon to the clinicians. Keywords: Acquired anomalies, Array-CGH, Constitutional disorders, Molecular karyotyping
Cytogenetic study has remained the clinicians’ favorite for many of the genetic disorders especially in unexplained psychomotor retardation [with/without dysmorphism], abnormality of sexual differentiation and development, infertility, recurrent pregnancy loss, pregnancy at risk for aneuploidy, chromosome breakage syndrome and cancer. However, limitation of band resolution has added the plethora of research studies in search for technology that can visualize chromosomal anomalies which are thought to be not existing or missed. This has transformed cytogenetics to the molecular cytogenetics with an unmatched precision and knowledge gained so far. Though chromosomal abnormalities remain a major cause of most of the genetic disorders, limitations of high resolution, missed many of the genetic disorders while molecular biology has its limitations of known targeted anomalies identification. These limitations have transformed the newer innovation with high end precision of cytogenetic techniques (Table 1). Nonetheless all these technology have their limitation that uses comprehensive analysis of the disorders —————— *Correspondent author’s present address: FRIGE (Foundation for Research in Genetics and Endocrinology), Institute of Human Genetics, FRIGE House, Jodhpur Road, Satellite, Ahmedabad 380 015, India Telephone: 079-26921414 Fax: 079-26921415 E-mail:
[email protected]
Table 1—Landmark in the history of conventional cytogenetic and molecular cytogenetic Year 1959 1969
1976 1981 1984 1986 1989 1989 1990 1990 1992 1993 1996 1996 1997 1997 1997 1998
Discovery First Chromosomal anomaly detected [Trisomy 21] Molecular hybridization of radioactive DNA to the DNA of cytological preparations High resolution banding technique Isotopic in situ hybridization Direct FISH Interphase FISH Combinatorial labeling Primed in situ hybridization [PRINS] Identification of translocation in Interphase cell by FISH Technique of ratio labeling Comparative Genomic Hybridization [CGH] Fiber FISH Multiplex FISH [M-FISH] SKY Cross species color Banding [Rx FISH] DNA array (matrix) CGH Padlock probe FISH Array CGH using BAC clones
Ref. 1
Lejune J
Pardue & Gall2
Yunis3 Harper & Saunders4 Landegent5 Cremer et al6 & Pinkel et al7 Nederlof et al8 Koch et al9 Tkachuk et al10 Nederlof et al11 Kallioniemi et al12 Parra & Windle13 Speicher et al14 Schrock et al15 Muller et al16 Solinas-Toldo17 Nilsson et al18 Pinkel et al19
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using cytogenetics to molecular cytogenetics to array—comparative genomic hybridization (CGH). Table 2 shows comparison of each technology, its uses and limitations for understanding genomic imbalances in various genetic disorders. Techniques and levels of resolution Conventional cytogenetic [CC] technique, the standard karyotype (400-bands per haploid genome) allows a level of resolution, from 10-15 Mb (million base pairs). In the best conditions, the high-resolution karyotype (550-bands) allows to reveal anomalies of about 3 to 5 Mb according to the regions of the genome and the technique used (G-bands, R-bands). The limitation of enhance resolution of the karyotype is not only linked to the cytogenetic technique used (it is possible to get karyotypes up to 850-bands), but also the region of interest. Secondly, the cytogenetic technique is only partly automated; need insight, indebt knowledge and experience of the cytogeneticists.
Molecular biology allows precise analysis of variations at the nucleotide level. Molecular cytogenetic techniques, mainly Fluorescence in situ hybridization (FISH), which is currently useful on both metaphase chromosomes and interphase cells, utilize genomic sequence-probes in bacterial artificial chromosome [BAC 100-200 kb] cloned in Escherichia coli or P1 artificial chromosome [PAC 100-150 kb] cloned in phage. These sequences are tagged with fluorochrome, which allows the detection of genomic imbalances involving chromosome segments smaller than 1 Mb for deletions and more than 2 Mb for duplications. This technique is more useful to detect micro-deletion and alteration at the telomeric region, which are not identifiable with conventional cytogenetics (CC). This can be confirmed and correlated with the clinical symptoms. It is important to know that this technique can identify only the specific region of the interest [specific sequence used as probe] and not the whole genome in a single assay.
Table 2—Comparison of technical details between conventional cytogenetic and major molecular approaches Method Conventional cytogenetics
Analysis
Resolution
Cell culturing: arresting cell in metaphase High resolution arresting cell in banding technique metaphase
Whole genome
10-15 Mb
Whole genome
3-5 Mb
Conventional FISH
Molecular biology technique
Identify specific region
Spectral Karyoytping/ MFISH
arresting cell in metaphase
Whole genome
1-2 Mb
CGH
Molecular Whole genome biology technique
3-5 Mb
Array-CGH
Molecular Whole genome biology technique
20 - 150 Kb
Applications
Limitation
Detection of balance and unbalance Minor rearrangements defects cannot be detected
Detection of balance and unbalance Micro-deletions and defects terminal aberrations can not be detected. Detect all types of balanced and Structural anomalies 1-3 Mb Metaphase unbalanced defects in metaphase, cannot be detected in noninterphase cell, frozen section and dividing cells. Smaller dead cells. FISH is highly sensitive 50 Kb for trisomy than Interphase cells monosomy Detect numerical and structural Inability to detect paracentromeric inversion, aberrations including subtle rearrangements, complex small deletion/duplications translocation, small marker, ring and cryptic translocation and double minute chromosomes in a single experiments with all 24 chromosomes No need for cell culture or Cannot detect Balanced metaphase, does not require fresh translocation, gain/loss of sample, frozen or fixed sample can
