Genetic Diagnostic Methods

Tissue Culture And Chromosome Analysis
Human somatic cells have 46 chromosomes consisting of 23 pairs. The 22 pairs, which are termed as autosomes, are the same between males and females. The remaining one pair constitutes the sex chromosomes (gonosomes) and is generally found as XX in females and XY in males. Chromosomes are the structures which contain the genetic code (genes) in the nucleus of cells. 
Chromosomes are isolated when viable, dividing nucleated cells are inhibited at the mitotic stage after cells proliferate in appropriate culture conditions. The karyotype analysis can be reported once isolated chromosomes are stained and investigated for alterations in numerical and structural characteristics.

The structural and numerical aberrations in chromosomes are associated with several syndromes and diseases and in particular observed in individuals who cannot menstruate, conceive a baby, experience repetitive spontaneous abortions or have physical examination reports of chromosome abnormalities such as Down syndrome and in patients diagnosed with leukemia. The gain or loss of a whole chromosome or its segment can result in unbalanced numerical chromosomal anomalies including mental retardation, anomalous baby births, spontaneous abortion and fertility problems. On the contrary, individuals with structural chromosomal anomalies such as translocation or inversion may not experience health problems or differences in learning ability due to the absence of genetic loss and therefore known as carriers.  However, the chromosomes transmitted to the babies of carrier individuals may have unbalanced genetic information which could result in anomalous births or spontaneous abortion. Therefore, it is important for these individuals to obtain genetic diagnosis by cytogenetic methods which support the analysis of all chromosomes or by molecular cytogenetics (FISH etc) to analyze specific chromosomal segments. Chromosome analysis is required following genetic counseling for patients who are at risk of having a genetic disorder or in the case of a known familial genetic disease. The peripheral blood, bone marrow, skin biopsy or abortion material represent various tissue samples to be used for the isolation of chromosomes.

Karyotype Analysis:
The karyotype analysis is frequently performed on cells isolated from peripheral blood of patients although the type of tissue samples to be analyzed may vary depending on the type of chromosomal disease or the age of the patient at diagnosis. Patients must give informed consent before their tissue samples are taken and processed for analysis. Each tissue sample requires different conditions for storage and shipment to the laboratory. Therefore, specific criteria have been set for sample admittance and rejection. The time to produce a diagnostic report following chromosome analysis may change depending on the tissue culture conditions of patient samples.

Chromosomes are not visible to the naked eye and can only be seen under the microscope when they are stained after isolation from proliferating cells in culture. They have different bright and dark horizontal bands which are numbered starting from the centromere (the point where short and long arms of the chromosome join) towards the opposite end. This type of chromosome analysis is also termed as karyotype analysis. If alterations of chromosomes are big enough, karyotype analysis can detect chromosomal aberrations (the gain or loss of chromosomal content) or rearrangements.
The results of cytogenetic and molecular cytogenetic tests may become difficult to interpret or in some cases may not reflect the real situation in patients. It is easier to detect numerical or big structural chromosomal anomalies with these methods but small structural aberrations and mosaicism may remain as undetected. These tests are designed for the specific risk factors and therefore only give information about the disease in question. They do not give any information about other diseases. Furthermore, different diseases or anomalies of genetic or other origin can be seen even in individuals with a normal karyotype.

Chromosome banding is used to detect chromosomal rearrangements of 5-10 mb in size by analyzing chromosome structure and number depending on differences in banding pattern. Fluorescence in situ hybridization (FISH) is a specific and sensitive method which detects localization of genes on the chromosomes. This technique can identify chromosomal abnormalities of 1-3 mb which cannot be normally visualized by light microscope. It is also possible to analyze numerical and/or structural anomalies in the nucleus of cells without prior isolation of chromosomes. Therefore, FISH can be used during preimplantation, prenatal or postnatal diagnosis of genetic diseases by investigating gene deletions, translocation and amplification.  

FISH is a method of molecular cytogenetics which is commonly used in cytogenetic diagnosis and involves analysis of chromosomes and nucleus isolated from cultured cells following hybridization with DNA probes tagged with fluorescent dyes. This method only gives information about the chromosome regions which are recognized by the DNA probes and therefore cannot identify abnormalities in other regions of the same or different chromosomes. 

aCGH (array Comparative Genomic Hybridization): aCGH is a new method which allows detailed investigation (25 kb resolution) of the genome. aCGH investigates DNA from patients and healthy individuals and compares differences between two samples. By this way, copy number imbalances (gain or loss) in the DNA can be identified. This method enables the analysis of unbalanced constitutive rearrangements in the genome and identifies their effects on the genes. The copy number imbalances are small alterations of the DNA which cannot be detected under the microscope and therefore termed as "submicroscopic alterations". They can have an effect on growth and development as well as on gene expression which contribute to the development of several diseases in adults.
In this respect, aCGH is used as a routine diagnostic test in our department to describe the gene or gene regions responsible for the etiology of a disease in patients with relevant diagnosis and whom cannot be investigated by other diagnostic methods.

The Working Principle of aCGH: Microarrays consist of thousands of DNA probes attached to a glass surface. Initially, the DNA molecule from a patient is digested into small fragments which are tagged by a fluorescent dye. DNA from individuals who have no known anomalies is also used as the reference DNA which is again digested into small fragments but tagged with a different fluorescent dye. Reference and patient DNA samples are mixed together and applied on the same slide. By this way, DNA fragments are allowed to hybridize to complementary probe sequences attached to the glass slide. Glass slides are then scanned by the microarray scanner which measures the fluorescent signals emitted by the patient and reference DNA fragments hybridized to the probes and thus detects deletion or duplication in the genome of the patient.

Molecular Genetic Tests

Heredity is the information which affects the characteristics of the progeny when transferred to the next generation. The universal characteristic of living organisms is to store, use and transfer the hereditary information to the next generation in order to fulfill their functions in life. Nucleic acids, which are the building blocks of the cells, are responsible for carrying out these functions.

Nucleic acids are made up of repeating units of nucleotides. Each nucleotide contains three parts: 1) A nitrogenous heterocyclic base, 2) Five-carbon sugar (pentose) and 3) A phosphate group. There are two nucleic acid molecules in a cell. The nucleic acid with a ribose sugar is called ribonucleic acid (RNA) and the nucleic acid with a deoxyribose sugar is called deoxyribonucleic acid (DNA). In addition, DNA and RNA molecules also differ by the type of nitrogenous base they contain: adenine, guanine and cytosine are found in both types of nucleic acids but thymine is only seen in DNA and uracil only in RNA. Most organisms, except some viruses, store hereditary information in their DNA molecules.

The scientific developments since the discovery of molecular structure of DNA by Watson and Crick in 1953 have not only shown how the flow of hereditary information inside a cell is achieved but also promoted the development of molecular biology techniques that can be used in the diagnosis of genetic diseases. 

Previously, diagnosis of genetic diseases has been restricted to clinical evaluation and biochemical tests based on protein analysis and therefore genetic diagnosis has not been widely used for several years. The main reason for this is the necessity to find the correct tissue that express the protein required for analysis since the expression of proteins can differ from tissue to tissue during cellular differentiation. The fact that DNA molecule is present in all nucleated cells and the relative ease of obtaining DNA from tissues such as peripheral blood have enabled the use of several diagnostic tests which are regarded as a milestone in molecular medicine and include "predictive" tests (to demonstrate if an individual has the risk for developing a certain disease) as well as "presymptomatic" diagnosis (shows if the disease causing gene(s) responsible for a familial inherited disease is present in an individual), "prenatal" diagnosis (before birth) and "preimplantation" diagnosis (shows predisposition of the embryo to a genetic disease before its transfer to the mother’s uterus).

Furthermore, with recent developments in scientific knowledge and technology it is now possible to analyze RNA samples for diagnosis and follow-up of specific diseases. The diagnostic tests based on nucleic acids (DNA and RNA), which have also become widely used applications in medicine, are very sensitive methods and provide important guidance in the diagnosis, prognosis and planning of relative treatment regimens for several diseases.

Tests In Use

Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR) Test: QF-PCR is used in the detection of prenatal chromosomal anomalies. The amniotic fluid obtained during amniocentesis performed by a gynecologist contains cells shed from the skin of the fetus which can be used as a source for DNA isolation. Specific regions in the chromosomes isolated from this DNA are amplified by fluorescent tagged chemicals. Fragment analysis is used to detect the increase in chromosome numbers using target chromosome specific markers.

Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP): The analysis of base substitutions which can result in mutations in the DNA requires the amplification of the DNA molecule. It is possible to amplify the specific regions of the DNA in a test tube using appropriate chemicals by polymerase chain reaction (PCR). The presence of base substitutions can change the recognition sites of the restriction enzymes in the target region. Therefore, the PCR amplified region of the DNA is subjected to restriction enzyme digestion and the resulting profile of fragments produced is analyzed in this test.

Reverse Hybridization

Reverse hybridization enables investigation of multiple regions of the DNA molecule simultaneously. This technique, which is also known as "Multiplex" PCR, involves amplification of several target regions at the same time in a single reaction using appropriate chemicals. In the following step, a specific test strip is used which contains wells filled with chemicals that can recognize healthy and mutant DNA fragments. PCR products are mixed with these chemicals and the resulting reaction in the wells is analyzed.

Real Time PCR

Real time PCR is based on simultaneous amplification of nucleic acids and the detection of increase in fluorescent signal. It is a technique commonly used in DNA analysis in addition to gene expression studies.

DNA Sequencing

DNA sequencing indentifies the sequence of nucleotides (adenine, guanine, cytosine, and thymine) in the DNA molecule. Initially, the region of the DNA molecule to be investigated is amplified using specific chemicals. These PCR products are purified and amplified once again using fluorescent tagged chemicals this time. Samples are separated by capillary electrophoresis and the fluorescence emission is captured and analyzed on the computer.

Inspection and Verification of Identity of Cell Lines
Cell lines used in research laboratories may become contaminated or lose some of their characteristics after exposure to repeated cycles of passaging. For this reason it is important to verify the identity of cell lines when they first arrive to the laboratory and also if they present unusual behavior during their culture.

Mycoplasma Test

Infections with mycoplasma interfere with the growth rate and the metabolism of cells in culture and can cause researchers to experience several problems. It is therefore critical to test cultures of cell lines for mycoplasma infection when cells first arrive to the laboratory or if any problems are encountered throughout their culture. 

Immortalization Of Lymphocytes

Peripheral blood lymphocytes can be used as a cell source for analysis in research and routine applications. However, more blood may be required from patients to isolate lymphocytes if there is a shortage of biological materials such as DNA samples. The immortalization of lymphocytes is an important application to provide continuum of biological materials in situations when it is not easy or possible to reach the blood donor.