Clinical applications of comparative genomic hybridization

Abstract

Purpose: Comparative genomic hybridization (CGH) is a powerful DNA-based cytogenetic technique that allows the entire genome to be scanned for chromosomal imbalances without requiring the sample material to be mitotically active. During the past 2 years we received many requests from various medical centers around the country to use CGH to resolve the identity of aberrant chromosomal material. Methods I M U C T I O NApproximately 3% to 4% of liveborn babies have a major congenital defect.' These defects include unbalanced chromosome abnormalities which occur in approximately 1 in 250 newborm2 Between 15% and 20% of all pregnancies end in a spontaneous abortion, and of these approximately 50% are associated with chromosome abn~rmalities.~ A precise diagnosis in a newborn or prenatal sample with a chromosome abnormality is critical for appropriate genetic counseling as well as for the clinical management of an infant. It also provides the parents with a realistic prognosis. There are many instances when cytogenetic analysis is unsuccessful in producing a result because the specimen cannot be cultured. There are also many occasions when extrachromosomal material remains unidentifiable even after numerous standard cytogenetic staining methods have been attempted. Molecular cytogenetic techniques, such as fluorescence in situ hybridization (FISH), have brought a new depth to clinical cytogenetics by facilitating the identification of chromosomal material of unknown origin. This approach often requires using multiple whole chromosome paints (WCP) until the source chromosome is identified.s5 Alternative approaches include reverse FISH, using probes derived from the microdissected chromosome region of interestM; and multicolor FISH p r~b i n g .~. 'O However, these techniques require specialized equipment in addition to the regular cytogenetic/FISH image analysis equipment that now is commonly found in many comprehensive cytogenetic laboratories. Although multicolor FISH can determine the origin of the unknown material, it does not identify the specific location or breakpoints on the chromosome from which the extra material originated.Comparative genomic hybridization (CGH) is a relatively new molecular cytogenetic technique that allows for the identification of chromosomal gains or losses by scanning the entire genome in a single step. It has the distinct advantage of providing a genome-wide search without any prior &formation about the chromosomal aberration in question. It is accomplished by in situ hybridization of differentially labeled total genornic specimen DNA and normal reference DNA to normal human metaphase chromosome ~preads.'l-~~ Hybridization of the specimen and reference DNA can be distinguished by their Merent fluorescent colors. The relative amounts of specimen and reference DNA hybridized at a particular chromosome position are contingent on the relative excess of those sequences in the two DNA samples and can be quantified by calculation of the ratio of their different fluorescent c~l o r s .~~-l~...

Characterization of the first supernumerary tricentric ring chromosome 1 mosaicism by conventional and molecular cytogenetic techniques

“…Even though only 8 % of the cells showed the presence of the marker chromosome, in which the content of the euchromatic part 1q21 is fivefold instead of twofold mimicking a 24 % mosaicism for partial trisomy 1q10q21, the aberration was detectable by CGH using narrow thresholds. So far, 19 cases of "pure" SMCs derived from chromosome 1 containing different pericentric euchromatic parts of 1p and/ or 1q have been described (Callen et al, 1990(Callen et al, , 1991(Callen et al, , 1999Michalski et al, 1993;Plattner et al, 1993;Chen et al, 1995;Lanphear et al, 1995;Crolla et al, 1998;Levy et al, 1998;Xu et al, 1998;Giardino et al, 1999;Anderlid et al, 2001;Dawson et al, 2001;Finelli et al, 2001;Röthlisberger et al, 2001;Tönnies et al, 2003). Regarding the size of the ring chromosome and the content of euchromatic material from proximal 1q, only four of the supernumerary ring 1 chromosomes reviewed in the literature are cytogenetically comparable (cytogenetically matching cases: Chen et al, 1995;Xu et al, 1998;Giardino et al, 1999;Finelli et al, 2001; see also Table 1).…”

“…Therefore, the precise identification and characterization of a marker chromosome and its euchromatic content is of essential importance in genetic counseling and diagnosis. So far, 19 cases of single SMCs derived from chromosome 1 containing different euchromatic parts of proximal 1p and/or 1q have been described (Callen et al, 1990(Callen et al, , 1991(Callen et al, , 1999Michalski et al, 1993;Plattner et al, 1993;Chen et al, 1995;Lanphear et al, 1995;Crolla et al, 1998;Levy et al, 1998;Xu et al, 1998;Giardino et al, 1999;Anderlid et al, 2001;Dawson et al, 2001;Finelli et al, 2001;Röthlisberger et al, 2001;Tönnies et al, 2003). We report here a 3.5-year-old girl referred for cytogenetic analysis because of language and motor delay who has a tricentric supernumerary ring chromosome 1 characterized by molecular cytogenetic techniques including YAC-FISH, chromosome microdissection followed by FISH, microsatellite analysis and CGH.…”

Prenatal molecular cytogenetic diagnosis of partial tetrasomy 10p due to neocentromere formation in an inversion duplication analphoid marker chromosome

“…CGH CGH probes were prepared and washed as previously described (Kallioniemi et al, 1994;Levy et al, 1998). CGH images were captured with an ImagePoint cooled CCD video camera (Photometrics) through a Labophot-2A fluorescence microscope (Nikon).…”

“…Chromosome identification and karyotyping were facilitated by counterstaining with 0.5 Ìg/ml DAPI in Vectashield (Vector Laboratories) (Florijn et al, 1995). CGH analysis was performed as described by Levy et al (1998). The fluorescence ratios (green/red) for at least 10 of each autosome and 7 of each sex chromosome were obtained per slide.…”

“…Reverse FISH (Ohta et al, 1993;Thangavelu et al, 1994;Viersbach et al, 1994), where the marker is microdissected, DOP-PCR amplified, and hybridized back to normal chromosomes, reveals both the origin and position of the marker, but it is a highly specialized, labor-intensive procedure not amenable to routine clinical application. Comparative genomic hybridization (CGH), where patient and normal reference genomic DNA are differentially labeled and hybridized to normal metaphase chromosomes, has been particularly useful in clinical cytogenetics and reveals both the origin and position of extra chromosomal material (Bryndorf et al, 1995;Levy et al, 1997;Levy et al, 1998).…”