Karyotyping from blood can be carried out for all age groups.
Indications for cytogenetic analysis:
- Dysmorphic features
- Developmental delay
- Congenital anomalies
- Ambiguous genitalia
- Hypogonadism / undescended testes / hypospadias
- Delayed menarche and short stature
Sample: 2-3 ml blood in sodium heparin vaccutainer (green top tube) transported at room temperature
Genetics is the study of heredity and genetic variation. It deals with hereditary diseases and birth defects.
Genetic Diagnostic Tests can be broadly classified into:
- Cytogenetic Tests – Karyotyping & FISH for chromosome analysis.
- Molecular Genetic Tests – PCR, sequencing, microarray and Next Generation Sequencing for DNA analysis.
- Biochemical Genetic Tests – To detect inborn errors of metabolism.
Chromosomes are X shaped thread like structures made up of DNA, and carry the hereditary material of an individual. Different genes are located at specific points on chromosomes, which are visible under the microscope when the nucleus of a cell is dividing.
Human beings have 46 chromosomes. These are present in pairs. This is termed as a diploid set of chromosomes. The sex chromosomes are XX in females and XY in males. The other chromosomes, besides the sex chromosomes are called autosomes. The ova and sperm however contain only 23 unpaired chromosomes each (a haploid set) so that when fertilization takes place, the cells of the embryo will again have 46 chromosomes.
Chromosome abnormalities are mainly of two types.
- Numerical – e.g. Trisomy, Monosomy, Triploidy, Tetraploidy, Mosaicism
- Structural – e.g. Translocation, Deletion, Inversion, Duplication
Trisomy is the most common type of numerical chromosome abnormality. There is one extra chromosome in any pair e.g. Trisomy 21. These individuals suffer from Down syndrome. Thus the total number of chromosomes in each cell will be 47 instead of 46.
Down Syndrome is a genetic disorder where the individual is usually mentally challenged. Such individuals have typical facial features like upslanting eyes, depressed nasal bridge, and an open mouth with a rough protruding tongue. They often have a single palmar crease or Simian crease on their palm. Trisomy 21, is the most common cause of Down syndrome.
Down syndrome is occasionally caused by a translocation when 2 chromosomes have fused, so the total number of chromosomes remains 46 instead of 47. This can be inherited from a parent who is a carrier, having 45 chromosomes instead of 46 because of this fusion. In such cases, there is a risk that subsequent children may also be affected. Prenatal Diagnosis is important in these cases to determine if the fetus has Down syndrome. In the rare instance of a parent carrying a 21/21 translocation, all the children will have Down syndrome, so assisted reproduction with donor sperm or oocytes can be offered accordingly. This illustrates the importance of Karyotyping.
Karyotyping is the process of chromosome analysis using banding techniques. The chromosomes seen under the microscope are arranged in pairs and scrutinized for any visible chromosome abnormalities. The method involves tissue culture to obtain dividing cells. Hence collection of appropriate samples under aseptic conditions is important.
- Turner Syndrome (45,X and variants)
- Klinefelter Syndrome (47,XXY and variants)
- Down Syndrome (Trisomy 21, translocation and mosaicism)
- Couples with infertility of unknown cause
- Couples with recurrent spontaneous miscarriages
- Children with ambiguous genitalia
- Female children with inguinal hernia
- Children with mental subnormality and dysmorphic features
- Suspected cases of Fanconi anaemia, Ataxia Telangiectasia, Bloom syndrome
- Bone marrow analysis in leukemias (blood cancer).
- Prenatal Diagnosis of fetal chromosome disorders in high-risk pregnancies.
Aneuploidy is the presence of one extra chromosome (trisomy) or absence of one chromosome (monosomy) in each cell. This leads to different abnormalities such as:
- Trisomy 21- Down syndrome or Mongolism
- Trisomy 13 – Patau syndrome
- Trisomy 18 – Edward syndrome.
- Sex chromosome abnormalities- XXX, XXY, XO, XYY.
In products of conception (tissue from spontaneous abortions) trisomy 16 and trisomy 22 are quite common.
Common aneuploidies can be rapidly detected by a cytogenetic technique called Fluorescence in situ hybridization (FISH).
FISH (Fluorescence in situ hybridization) is a rapid molecular cytogenetic technique. FISH is mainly used to detect common aneuploidies like trisomy or monosomy of chromosomes 21, 18 or 13, sex chromosome abnormalities like Turner (XO) or Klinefelter (XXY) syndrome and mosaicism where there is a mixture of normal and abnormal cells. This test is important in a variety of cases ranging from prenatal diagnosis to cancer. The test results are available in 1-2 days as tissue culture is not required. There is no risk of a culture failure due to contamination or inadequate sample size.
The FISH test has many benefits. It reduces parental anxiety especially in prenatal diagnosis when the Triple test shows a high-risk pregnancy, as the FISH report is available much earlier than the karyotype reports. A large number of interphase nuclei can be studied to detect low-grade mosaicism. The test can also be carried out on a wide range of samples like chorionic villi, amniotic fluid, cord/adult blood, placental biopsy, products of conception, buccal cells and sperm. FISH has wide applications in detecting the type of cancer and in the prognosis of the disease.
Mosaicism is the presence of chromosomally normal and abnormal cells in a person. In such cases, the clinical manifestation varies according to the percentage of normal and abnormal cells. Low-grade mosaicism, where the percentage of one of the cell-lines is very small, can be easily detected by FISH.
Microdeletions are deletions of very small segments of chromosomes. They are often missed by karyotyping, but can be easily detected by FISH. Prader-Willi /Angelman Syndromes may be caused by a microdeletion on chromosome 15.
Commercially available DNA probes labeled with different fluorescent dyes are hybridized to the nuclei of cells, and analyzed under a fluorescence microscope. If the probe for chromosome 21 is labeled with an orange fluorescent dye, for example, we will see 2 orange signals under a fluorescence microscope in normal cells, and 3 orange signals in cells of an individual with Down syndrome (Trisomy 21). The diagnosis is thus made by counting the number of signals of different colours in each cell. The cells are counterstained with a blue dye DAPI, to differentiate the cells.
All chromosome abnormalities cannot be ruled out by FISH. Hence it cannot replace conventional karyotyping. FISH is useful to detect probe-specific abnormalities only.
FISH is commonly used in the rapid diagnosis, prognosis and management of chronic myeloid leukemia (CML) and acute promyelocytic leukemia (AML-M3), especially since specific treatment is available in each case. FISH is also used in many other hematological malignancies such as ALL, AML, MDS, Multiple Myeloma, CLL, Lymphomas and in solid tumors.
Prenatal diagnosis is the detection of certain cytogenetic, molecular or biochemical genetic abnormalities in the unborn child.
Prenatal diagnosis is recommended in the following cases:
- Advanced maternal age
- Screening tests show high risk of abnormality
- Abnormalities on ultrasonography
- A parent with a balanced translocation
- History of a previous abnormal child
- X-linked genetic disorders
- Couples with Thalassemia trait
- Couples with family history of known monogenic disorders such as Beta Thalassemia
- Cases in which NIPT (Non-Invasive Prenatal Testing) show a high risk for Trisomy 13, 18, 21.
Prenatal diagnosis can be carried out in the 1st, 2nd or 3rd trimester depending on the stage at which an abnormality is detected or suspected. Different tissues are sampled according to the gestational age, e.g.
- Chorionic villus (which forms the placenta) at 10-12 weeks
- Amniotic fluid (which surrounds the fetus) at 16-18 weeks
- Cord blood (from the umbilical cord) at greater than 19 weeks.
As per the PCPNDT (Prohibition of sex selection) Act 2003, the sample collection and analysis has to be carried out in registered clinics/ laboratories/centers as applicable after the mother has signed an informed consent form. The sex of the fetus is not revealed.
PCR (Polymerase Chain Reaction) is a molecular diagnostic technique used to generate (amplify) large amounts of specific sequences of genes from DNA, for genetic analysis. It can be used to diagnose single gene disorders like beta-Thalassemia and Cystic Fibrosis, Sickle Cell Anaemia, Duchenne Muscular Dystrophy, Metabolic Disorder in the new born such as Phenyl Ketonuria and Familial Hereditary Cancer Syndromes.
There are small regions on the Y chromosomes which are responsible for mature sperm formation. If a part or parts of the region is lost, it is noted as a deletion. These regions on the Y chromosome are too small to be detected through the microscope. These deletions can be detected by multiplex PCR technique. They are usually seen in males with azoospermia (No sperm found in the semen sample). Depending on which region is deleted, men with these deletions can father a child with Assisted Reproduction Techniques, but the couple has to be counselled that the male partner may transmit infertility to the male offspring.
PGT is Preimplantation Genetic Testing. It is an additional step during the ICSI procedure, where trophectoderm cells from an embryo can be tested to rule out certain genetic conditions such as chromosomal aneuploidy, thus allowing only normal embryos to be transferred.
During Genetic Counseling, the inheritance pattern of a particular genetic disorder in a family is studied. The chances of recurrence of the same abnormality in the family are explained. If genetic tests for diagnosis of that disorder are available, arrangements are made to send the required samples for testing. Prenatal diagnosis in a subsequent pregnancy in family members ‘at risk’ can be carried out. As most genetic disorders cannot be cured, genetic counseling helps to prevent the recurrence of the same disorder in the extended family.