Methods of Molecular Cytogenetics and Cytogenomics

More about Molecular Cytogenetics Methods

FISH

FISH is the basic method of molecular cytogenetics is in situ hybridization, which is used in various methodological variations.

This method allows to visualize nucleic acid sequences directly on microscopic slides containing morphologically preserved chromosomes or cell nuclei. Molecular in situ hybridization is based on the use of a labelled single-stranded hybridization probe, i.e. a short sequence of DNA. This probe can bind to the target DNA sequence of cells on a microscope slide after denaturation due to base complementarity. Depending on where the DNA probes hybridize, they are classified as cetromeric, whole-chromosome and gene-specific.

Currently, the most commonly used probes are approximately 200-300 kb in size and are conjugated to some of the fluorochromes (e.g. SpectrumGreen, SpectrumOrange), hence this technique is referred to as fluorescence in situ hybridization (FISH). The sites where the probe has bound can be identified using a fluorescence microscope. The number and position of individual fluorescent signals then informs us about possible numerical and structural changes in the chromosomes. Depending on the number of target sequences that can be simultaneously detected on a single slide, we distinguish between single-color, double-color or multi-color fluorescence in situ hybridization.

MLPA

This technique is nowadays standard method of molecular cytogenetics, which has been used at OLG FN Brno since 2006. The principle of this method is to bind oligonucleotide probes to the target DNA sequence based on their complementarity. Each probe consists of two oligonucleotides. After hybridization to the target site, the oligonucleotides are joined by ligation. After denaturation, only the linked probes are amplified by a conventional PCR reaction using a single primer pair. The amplified probes are separated by capillary electrophoresis based on their different lengths. Finally, they are analyzed using a special computer program.

The advantage of this method is that up to 40 different stretches of DNA can be examined at the same time using one MLPA reaction kit. It is possible to test a large number of DNA samples simultaneously, which is very efficient in routine diagnostics. It is a highly sensitive technique and can detect sequences differing in a single nucleotide. Only 20ng of DNA is needed per MLPA reaction, which can be of different origin (isolated from peripheral blood, amniotic fluid, tumor tissue...). The MLPA method can only detect numerical changes such as deletions or duplications, but does not detect balanced rearrangements such as translocations or inversions. Disadvantages include high sensitivity to contamination.

Currently, 6 MLPA reaction kits are used in our laboratory. P250 kit for the detection of DiGeoge syndrome, P245 and P297 kits for microdeletion screening, P070 and P036 kits for subtelomeric screening of patients with mental retardation, and methylation-specific kit for syndrome differentiation: Beckwith-Wiedemann syndrome (BWS) Russell-Silver syndrome (RSS) number ME030.

array-CGH 

The array-CGH technique is primarily designed to search for deletions and duplications in the genome of patients. It was developed by combining the advantages of classical CGH and DNA microarrays and could be characterized as CGH using short DNA fragments attached to a slide instead of metaphase chromosomes. These can be oligonucleotides, BACs, PACs or cDNA clones. The size and distribution of the attached fragments determines the resolution of the microchip type, which can range from tens of bases to kilobases. Overall, however, it is orders of magnitude higher than that of conventional CGH, which takes cytogenetic diagnostics to the molecular level.

The array-CGH procedure is based on the same principle as the CGH technique. The DNA to be examined and the reference DNA is isolated from patient and healthy donor cells, respectively, and labelled with color-coded fluorochromes. The labelled DNA is mixed in an equimolar ratio and hybridized to oligonucleotide fragments fixed on a chip. Hybridization is followed by washing of unbound probes and evaluation with a special scanning device - a scanner. This takes a laser image of the chip in both green and red.

Specially designed software then automatically identifies each spot on the chip and determines the ratio of the two fluorochromes. If the log2 of the normalized ratio of the fluorochromes is greater than 0, we speak of gains, otherwise we speak of losses of genetic material. The next step is then to assign each spot to a specific position in the genome, thus making the changes on the chromosomes visible. Array-CGH enables genome-wide screening of unbalanced changes with high resolution. Its disadvantage remains the inability to detect balanced changes (balanced translocations or inversions).

MPS (Massive parallel sequencing)

The "next generation", massive parallel sequencing (MPS) method is primarily designed to search for changes in the DNA sequence in the genome of patients.

The aim of this analysis is to detect changes in DNA sequence in selected regions (targeted MPS), in coding regions of the genome (whole exome sequencing- WES) or within the whole genome (whole-genome sequencing - WGS).

In our laboratory, it represents the next step in the investigative algorithm of pediatric patients with idiopathic mental retardation and congenital developmental defects (MR/CD) in whom we have not detected the genetic cause of their pathological phenotype using available methods (karyotyping, FISH, MLPA, array-CGH...). The DNA of the examined individuals undergoes several processing steps resulting in a library of DNA fragments that represent the targets for further analysis. The DNA fragment libraries are then sequenced on high throughput sequencers and the subsequent output in the form of raw data is processed using advanced bioinformatics algorithms. The output of these analyses is a set of variations in the patient's DNA sequence compared to the reference DNA, and these variants are subsequently filtered and analyzed in relation to the patient's phenotype.

For the purpose of this investigation, we simultaneously offer two approaches: targeted NGS of the patient's DNA followed by verification of variants of clinical significance in the patient's parents using Sanger sequencing, or WES based "trio" analysis, where the DNA of the patient and his/her parents undergo the entire process simultaneously.

The MPS technique represents an important step in the diagnostic algorithm of our laboratory, where its output appropriately complements the results of previous analyses. Its main advantage lies in the identification of pathogenic sequence variants, which, according to the available literature, may be the cause of the pathological phenotype in more than 50% of paediatric patients with MR/VVV. The main limitations remain in the advanced complex bioinformatic processing of the underlying sequencing data as well as in their subsequent interpretation.