The Application Of Comparatative Genomic Hybridization In Detection Of Genomic Imbalances In Fetuses With Malformations And In Prenatal Diagnosis

The application of comparative genomic hybridization in detection of genomic imbalances in fetuses with malformationsBackgrounds: Congenital anomalies are present in approximately 10% of newborn infants. Potentially lethal or handicapping major defects occur in 2% - 3% of liveborn infants and have become the main cause of infant mortality during the first years of life. Genomic imbalances are a significant component of their etiology. Currently, conventional cytogenetic analysis of metaphase chromosomes and fluorescence in situ hybridization (FISH) are the most common methods for screening for abnormalities. These methods are very powerful, but have significant limitations. Standard cytogenetics may cause errors due to external contamination, culture failure and selective growth of maternal cells. The probes used in FISH technology are not designed for prenatal detection of all unbalanced chromosomal abnormalities. Only a few regions of the genome can be explored in one preparation. In 1992, Kallioniemi et al were the first to publish CGH as a new chromosome analysis technique which made quite a progress in cytogenetic approaches. In a typical CGH analysis, genomic DNA from the cells of interest (test DNA) and genomic DNA from karyotypically normal cells (reference DNA) are respectively labelled with a green and a red fluorochrome. Then, test and reference DNA are cohybridized as probe in equal amounts to normal metaphase spreads with unlabeled Cot-1 DNA to suppress hybridization of interspersed repeated sequences. Images of the fluorescent signals are captured, and the green-to-red signal ratios are digitally quantified for each chromosome homologue. Chromosomal locations of copy number changes within the DNA segments of the test genome are revealed by a variable fluorescence intensity ratio (FR) along each target chromosome. The CGH technique has many advantages, since tissue that is fresh, frozen, formalin-fixed, or paraffin-embedded can be used for the analysis and the tissue or cells needed are much less. Comparative genomic hybridization is a technique that offers a molecular approach to cytogenetic analysis and allows the entire genotype to be screened in a single hybridization without the need of specific probe and prior knowledge of the location of chromosomal abberations. This technique can readily identify numerical and unbalanced structural chromosomal abnormalities and be efficient for precise localization. As a new and effective cytogenetic approach, CGH has been applied primarily in cancer genetics, but is considered useful for clinical cytogenetics as well, including the application in the research of malignant hematological diseases, cell lines, diagnosis of hereditary diseases and embryo development research. CGH can be used to detect all diseases caused by genetic gains or losses and be used as an assistant approach to the clinical cytogenetics.Objective: To evaluate the feasibility and reliability of comparative genomic hybridization (CGH) in the detection of genomic imbalances in malformed fetuses or infants diagnosed by ultrasound prenatally or after birth. Methods: Fetuses were chosen from medically terminated pregnancies (n-5) and pregnancies which had ended in spontaneous fetal death (n=l), and malformed infants (n=3) at the Department of Obstetrics of Shandong Provincial Hospital in Jinan, China from July, 2006 to January, 2007. All fetuses presented at least two anomalies, identified by ultrasound examination or after birth, in the cardiovascular, skeletal, urogenital, or central nervous systems. Either 5-10 ml amniotic fluid or 2-5 ml umbilical cord blood was obtained for these studies. A healthy man and woman were chosen as control group from the healthy investigation centre in Shandong Provincial Hospital. 2ml blood was obtained and stored in an EDTA containing tube. Genomic DNA was extracted from amniotic fluid or umbilical cord blood samples using a Tiangen DNA extraction kit according to the manufacturer's protocol. DNA concentration and purity was determined by DU^? series 600 spectrophotometer. Sample DNA and control DNA were labeled with SpectrumGreen dUTP and SpectrumRed dUTP respectively using the Vysis nick translation kit according to the supplier' s recommendation. The reaction was performed for 0. 5 to 1 h at 15℃to obtain a fragment length of 300-3000bp. Probes were prepared by mixing 200ng of SpectrumGreen dUTP labeled sample DNA, 200ng SpectrumRed labeled male human genomic DNA and 10μg of unlabeled Human Cot-1 DNA. The probe mixture was precipitated in sodium acetate and ethanol and resuspended in CGH hybridization buffer. The probe mix was added to the hybridization site on the normal male metaphase target slide and covered with a sealing film. The slide and probe mix were co-denatured at 73℃for 5 min and placed in a moist chamber at 37℃for 48-72 h. Following hybridization slides were washed in 0. 4×SSC/0. 3% NP-40 solution at 74℃for 3-6 s, followed by 2×SSC/0.1% NP-40 solution at room temperature for 1-3 s. Target chromosomes were then counterstained with DAPI II. Because conventional cytogenetic analyses of all cases were unavailable, we designed fluorochrome-exchanged CGH to verify our fingdings: Sample DNA was labeled with SpectrumRed dUTP and genomic DNA was labeled with SpectrumGreen. The protocol was otherwise identical to the CGH protocol outlined above. CGH slides were analyzed on an epifluorescence microscope equipped with a CCD camera using specific filter sets for DAPI, SpectrumGreen and SpectrumRed signals. For each sample, 5 to 10 metaphase spreads with high uniform hybridization and fluorescence intensity were chosen for image analysis. A sequence of blue, green and red digital images was acquired under VideoTesT CGH software control. Karyotyping was performed based on DAPI banding pattern. A fluorescence intensity ratio (FR) profile was calculated after background correction and normalization of the green to red ratio for each metaphase to 1. 0. Mean ratio profiles for each chromosome were determined after data from all analyzed metaphases were combined. Trisomies or partial chromosome gains were defined as FR>1. 25. Monosomies or partial chromosome losses were defined as FR<0. 75.Results: Each of the 9 samples was analyzed successfully for numerical chromosome aberrations by CGH. Numerical chromosome aberrations were identified by CGH in three cases, 4, 8 and 9, and were verified by fluorochrome-exchanged CGH. For case 4, trisomy 21 was detected by CGH (when sample DNA was labeled green), but by fluorochrome-exchanged CGH, only trisomy 21q was detected. Because of the prevalence of heterochromatic DNA, 21p was excluded from analysis. Human Cot-1 DNA suppressed the hybridization of labeled DNA to centromeric regions of acrocentric chromosomes and chromosomal regions with high concentrations of repeat sequences, FR profiles deviated from the diagnostic ranges and had to be discounted during CGH interpretation. For case 8, deletion 2p24-pter and duplication 12pl3 were identified by CGH and verified by fluorochrome-exchanged CGH. For case 9, the unbalanced karyotype of del 1p33-pter and del 22q11-12 was identified, although 22p had to be excluded from analysis because of heterochromatic DNA. Among the remaining cases, mean ratio profiles obtained by CGH indicated balanced karyotypes.Conclusions: Exchanging the labeled fluorochrome can reduce inconsistencies in the results caused by deviations in the process of DNA labeling and hybridization, and can increase the accuracy and reliability of analysis in cases where conventional cytogenetic analysis is unavailable. CGH can provide a safe and accurate alternative to traditional banding analysis, at least in the detection of aneuploidy. The application of short tandem repeat and comparative genomic hybridization in prenatal diagnosis of Down syndromeBackground: An extra copy of chromosome 21, i. e. trisomy 21, gives rise to Down syndrome, which is the most well known chromosome disorder. Persons with Down syndrome exhibit special characteristics as mental retardation which can vary from mild to moderate. Other characteristics are short limbs, heart defects and malformations of the gastrointestinal tract. Down syndrome occurs in about 1 in 800 live births, contributes to about 50% of congenital low intelligence, and is the most common reason of chromosomal disease causing congenital anomalies. There is no efficient therapy method, the only way is to diagnose early and selectively terminate pregnancy to avoid Down syndrome births. Screening tests of Down syndrome are non-invasive, and include maternal blood test and ultrasonographic screening. Deviations of the examined parameters from the median of normal pregnancies are converted into a risk factor by the combination assessment of maternal age, biochemical markers and ultrasound screening. If the risk is high enough to justify an invasive test, amniocentesis or chorionic villus sampling (CVS) is offered. Conventional cytogenetics using Giemsa banding of metaphase chromosomes has proven to detect a wide range of abnormalities with high trustworthiness, deserving the position of a gold standard in prenatal diagnosis. STRs are present all over the genome and constitute of short sequences of nucleotides that are repeated from a few to several thousands times thus forming different alleles of the STR. In general, it is always wise to use several different STR markers for each chromosome since it reduces the rate of misdiagnosis. The advantage of STR-PCR is the reduction of time, thereby reducing parental anxiety. Compared to the other mentioned methods, STR-PCR needs less material to determine an accurate result in a rather short time allowing a large number of samples to be processed simultaneously. It is also a labor-intensive and cost-effective method for prenatal diagnosis suitable for automation.Objective: In this study we employed multiple human chromosome 21 specific short tandem repeat (STR) DNA markers to determine the numbers of chromosome 21 present in fetal cells from high risk pregnancies. Positive fetuses were tested by comparative genomic hybridization to detect chromosomal aneuploidy. Compared to the conventional cytogenetic analysis, the specificity and reliability of this method can be determined.Methods: 79 cases of pregnant women were chosen who displayed high risk ratios in the screening of Down syndrome and need to undergo amniocentesis to get a definite diagnosis. 9 cases fetuses or infants with malformations were also selected. 28-30ml amniotic fluid was taken by amniocentesis from the high risk pregnant women, 25ml amniotic fluid was used for the conventional cytogenetic analysis, and the rest for STR-PCR. Amniotic fluid or umbilical blood was taken from malformed fetuses or infants without karyotyping. Genomic DNA was extracted using a Tiangen DNA extraction kit according to the manufacturer' s protocol. DNA concentration and purity was determined by DU^? series 600 spectrophotometer. Seven chromosome 21 specific tetra-nucleotide STR markers were chosen to detect the number of chromosome 21. Different fragments of amelogenin were amplified on chromosome X and Y which can determine the fetal sex. PCR was performed in 8 Eppendorf tubes under the same condition. Each 20μl reaction consists of 2μl of 10×Buffer, 1.6μl of 2. 5 mM dNTP, 2μl of 5μM forward and reverse primers, 0.1μl of Hot-Start Taq DNA polymerase, nμl of DNA templates (0.05μg), (12. 3-n)μl of ddH₂O. The thermo-cycling conditions were as follows: 94℃ for 3 min, 5 cycles of 94℃for 30s, 58℃for 30s, 72℃for 30s, and 30 cycles of 94℃for 30s, 52℃for 30s, 72℃for 30s, with a final extension of 72℃for 5 min. PCR products of the 8th tube were used to detect fetal sex by 1. 5% agarose gel electrophoresis. PCR products of the 1^st to 7^th tubes were used to detect the number of chromosome 21 by 10% polyacrylamide gel electrophoresis. The results were compared to the conventional cytogenetic analysis. Trisomy 21 fetuses were tested by comparative genomic hybridization to detect chromosomal aneuploidy.Results: DNA concentration extracted from 3-5ml amniotic fluid was 0. 0386±0. 0139μg/μl with the minimum of .0. 0225μg/μl, i.e., the minimum amount of DNA was 1. 125μg which was enough for PCR and CGH. DNA amount extracted from malformed fetuses or infants varied from 4-15. 65μg. There were 39 male fetuses and 49 female fetuses of the 88 samples. Two cases of Down syndrome were found. Case 35 displayed triple fragments of D21S1413 and D21S2039, whose karyotype according to CGH was Dup 21 and conventional cytogenetic analysis showed the karyotype of 47, XY, +21. Case X4 exhibited triple fragments of D21S1437, D21S2039 and D21S11, whose karyotype according to CGH and fluorochrome-exchanged CGH was Dup 21q without conventional cytogenetic analysis.Conclusions: The combination of short tandem repeat polymerase chain reaction and polyacrylamide gel electrophoresis can be performed rapidly and effectively in prenatal diagnosis of Down syndrome. Comparative genomic hybridization has advantages in aneuploidy detection.