Center for Molecular Biology and Genetics


Prenatal diagnostics in Karyo

  • nov 08, 2013



Preimplantation genetic diagnosis

Pre-implantation genetic diagnosis (PGD or PIGD) refers to genetic profiling of embryos prior to implantation (as a form of embryo profiling), and sometimes even of oocytes prior to fertilization. PGD is considered in a similar fashion to prenatal diagnosis. When used to screen for a specific genetic disease, its main advantage is that it avoids selective pregnancy termination as the method makes it highly likely that the baby will be free of the disease under consideration. PGD thus is an adjunct to assisted reproductive technology, and requires in vitro fertilization (IVF) to obtain oocytes or embryos for evaluation.

The term pre-implantation genetic screening (PGS) is used to denote procedures that do not look for a specific disease but use PGD techniques to identify embryos at risk. PGD is a poorly chosen phrase because, in medicine, to "diagnose" means to identify an illness or determine its cause. An oocyte or early-stage embryo has no symptoms of disease. They are not ill. Rather, they may have a genetic condition that could lead to disease. To "screen" means to test for anatomical, physiological, or genetic conditions in the absence of symptoms of disease. So both PGD and PGS should be referred to as types of embryo screening.

The procedures may also be called preimplantation genetic profiling to adapt to the fact that they are sometimes used on oocytes or embryos prior to implantation for other reasons than diagnosis or screening.

Procedures performed on sex cells before fertilization may instead be referred to as methods of oocyte selection or sperm selection, although the methods and aims partly overlap with PGD.


Microarray CGH

Microarray CGH enables copy number imbalance to be screened at a much higher resolution than is possible using traditional metaphase techniques. The ability to reliably identify small scale imbalances combined with greater automation, reduced subjectivity and removal of cell culturing have driven the adoption to array comparative genomic hybridization (arrayCGH) as a front line test for the investigation of developmental delay and other constitutional conditions.

Array comparative genomic hybridization (arrayCGH) has become a valuable, genome-wide screening tool for the detection of chromosomal aberrations in the form of copy number imbalances in the field of cytogenetics.

ArrayCGH is a molecular technique based on the co-hybridization of sample and control genomic DNAs. DNA extracted from a sample is fluorescently labeled and co-hybridized with normal control DNA to mapped DNA sequences that are spotted onto a glass slide surface. These clones contain DNA which represent the human DNA sequences, and are regularly spaced across the whole genome.

The major advantage of arrayCGH is that it is a genome wide screen at vastly improved resolution compared to traditional techniques. The resolution of detection is determined by the genomic distance between DNA clones, the number of clones on a microarray and the DNA length of the clone spotted on the microarray. Additionally, since only a DNA sample is required much of the required skill of traditional banding karyotyping and cell culturing is removed.


Karyotyping (G-Banding)

A karyotype (Greek karyon = kernel, seed or nucleus) is the number and appearance of chromosomes in the nucleus of a eukaryotic cell. The term is also used for the complete set of chromosomes in a species, or an individual organism.

Karyotypes describe the number of chromosomes, and what they look like under a light microscope. Attention is paid to their length, the position of the centromeres, banding pattern, any differences between the sex chromosomes, and any other physical characteristics. The preparation and study of karyotypes is part of cytogenetics.

The study of whole sets of chromosomes is sometimes known as karyology. The chromosomes are depicted (by rearranging a microphotograph) in a standard format known as a karyogram or idiogram: in pairs, ordered by size and position of centromere for chromosomes of the same size.

The basic number of chromosomes in the somatic cells of an individual or a species is called the somatic number and is designated 2n. Thus, in humans 2n = 46. In the germ-line (the sex cells) the chromosome number is n (humans: n = 23).

So, in normal diploid organisms, autosomal chromosomes are present in two copies. There may, or may not, be sex chromosomes. Polyploid cells have multiple copies of chromosomes and haploid cells have single copies.

The study of karyotypes is important for cell biology and genetics, and the results may be used in evolutionary biology (karyosystematics) and medicine. Karyotypes can be used for many purposes; such as to study chromosomal aberrations, cellular function, taxonomic relationships, and to gather information about past evolutionary events.

G-banding or Giemsa banding is a technique used in cytogenetics to produce a visible karyotype by staining condensed chromosomes. It is useful for identifying genetic diseases through the photographic representation of the entire chromosome complement. The metaphase chromosomes are treated with trypsin (to partially digest the chromosome) and stained with Giemsa. Dark bands that take up the stain are strongly A,T rich (gene poor). The reverse of G‑bands is obtained in R‑banding. Banding can be used to identify chromosomal abnormalities, such as translocations, because there is a unique pattern of light and dark bands for each chromosome.

It is difficult to identify and group chromosomes based on simple staining because the uniform color of the structures makes it difficult to differentiate between the different chromosomes. Therefore, techniques like G‑banding were developed that made "bands" appear on the chromosomes. These bands were the same in appearance on the homologous chromosomes, thus, identification became easier and more accurate. The acid-saline-Giemsa protocol reveals G‑bands.