Molecular Diagnosis of Genetic Diseases

An individual’s genetic composition plays an important role in susceptibility to all diseases. In other words, all diseases have a genetic component to them. But much more needs to be learnt in terms of the degree to which these genes contribute to diseases. Molecular tests can be employed to confirm a diagnosis in case of a symptomatic individual. And these tests can also be employed for prenatal diagnostic technique as they can detect changes in the genes and chromosomes of a fetus. These tests are offered to parents who have an increased risk of having a baby with chromosomal or genetic disorder. Molecular diagnosis basically looks for alterations or mutations in the DNA sequence of an individual.

A genetic disease, in simplest terms, can be defined as a disease caused by an abnormality in an individual’s genes which are contained within their genome. About 6000 genetic disorders are known until now. Many of these diseases are fatal or can cause severe health issues, while others may not necessarily cause health issues but can be triggered by non-genetic factors such as environmental factors, lifestyle habits, etc. Genetic disorders range from a defect in single base mutation in the DNA of one gene to chromosomal abnormalities that involve deletion, addition or substitution of chromosomes or sets of chromosomes. Genetic disorders can be hereditary, i.e., can be inherited by offspring from parents and vice versa. Genetic disorders can also be acquired due to certain changes or mutation in an individual’s DNA during their lifetime. Some of the causes for mutation are radiation and carcinogenic chemicals. There are four main types of genetic diseases:

  1. Single gene inheritance diseases: These diseases are caused by mutations or changes in the DNA sequence of a single gene. Examples are Marfan syndrome, sickle cell anemia, cystic fibrosis, hemochromatosis, and Huntington’s disease. These kinds of disorders are inherited in recognizable patterns such as autosomal dominant, autosomal recessive, and X-linked.

2. Multifactorial inheritance: This kind of inheritance occurs via a combination of environmental factors and mutations in multiple genes. This kind of inheritance is also called as complex or polygenic inheritance. For example, different genes that are known to influence breast cancer susceptibility are found on chromosomes 6, 11, 13, 14, 15, 17, and 22. Some multifactorial disorders are chronic diseases. Examples are cancer, obesity, high blood pressure, arthritis, Alzheimer’s disease, and heart diseases. Multifactorial or complex inheritance is also linked with heritable traits such as height, fingerprint patterns, skin color and eye color.

3. Chromosomal abnormalities: Chromosomes are located in the nucleus of each cell, and they are made up of DNA and protein. Chromosome aberrations can result in genetic diseases. Examples are Down’s syndrome, Turner syndrome, Klinefelter syndrome, etc.

4. Mitochondrial inheritance: This type of genetic disorder is caused by mutations in the non-chromosomal DNA of mitochondria. Mitochondria are round or rod like, small organelles that play a role in cellular respiration and are found in the cytoplasm of plant and animal cells. 5 to 10 pieces of DNA may exist within each mitochondrion. Examples of diseases caused by mitochondrial inheritance are Leber’s hereditary optic atrophy, Myoclonus epilepsy, a form of dementia called MELAS, which stands for mitochondrial encephalopathy, lactic acidosis, and stroke like episodes.

With the advances in molecular diagnosis of genetic diseases over the years, it has now become possible to detect these diseases as early as possible and even during the neonatal and prenatal stages of life. Three main types of tests are carried out for detecting genetic diseases. They are biochemical testing, cytogenetic testing, and molecular testing.

Prenatal testing: As the name suggests, prenatal testing is done before birth to detect changes in an unborn baby’s genes. Some techniques to obtain samples for fetal testing are described as follows:

  1. Amniocentesis: If done around the 12th week of gestation, the amniocentesis is early. Several prenatal diagnostic clinics perform amniocentesis between the 14th and 16th week of pregnancy. Studies have shown an increased loss of amniotic fluid if the amniocentesis is done prior to the 12th week and there is a risk of skeletal anomalies. Depending on the age of pregnancy, 10 ml to 30 ml of fluid is obtained during the procedure. Fetal cells from the skin, membranes, urinary tract, and digestive system are found in the fluid and recuperated by centrifugation of the specimen. These cells are then kept in culture for a period of 5 to 10 days in a culture medium to which calf serum has been added. Cellular multiplication is then sufficient and allows the preparation of microscopic slides allowing the numerical and structural studies of the metaphasic chromosomes. Treatment of chromosomes during the slide preparation of microscopic slides reveals segments of different intensity or bonding patterns. Those bands reflect a variable ratio AT and GC nucleotides on the chromatids and help in identifying chromosome pairs. But this method of sample collection can also be used in synergy with a rapid screening test for numerical aberrations of specific chromosomes, which can be used on uncultured amniotic fluid in conjugation with conventional cytogenetic analysis. Using FISH analysis (fluorescent in situ hybridization) with chromosome-specific probes at interphase nuclei or via molecular genetic analysis of highly polymorphic markers on a DNA probe isolated from uncultured amniotic fluid cells, information can be obtained regarding numerical abnormalities of chromosomes 13, 18 and 21, and the X and Y chromosomes. This test allows detection of the commonest chromosomal anomalies within one to three days. This test is of particular importance where morphological abnormalities potentially associated with the above conditions have been detected, and where a rapid diagnosis is required at a late stage of pregnancy. A prenatal rapid test can serve to reassure the pregnant woman if the child is normal, but this cannot replace formal karyotyping.
  2. Chorionic villus sampling (CVS): The biopsy or aspiration of chorionic villi by the vaginal route yields fetal cells, several of which are in the process of dividing and can be analyzed during the hours following the procedure. Chorionic virus sampling should not be carried out before the 11th or 12th week, because increased risk of limb abnormalities has been linked to carrying out CVS prior to the 11th or 12th week. There is also a risk of miscarriage and maternal cell contamination of the specimen. Depending on placental site, CVS can be carried out either trans cervically or transabdominally. Chromosome analysis is carried out as a direct preparation on following brief culture i.e., one day, as well as full culture i.e., 7 to 10 days. In special circumstances when the risk of genetic disease is high as for instance in hereditary metabolic diseases or if one of the parents is carrier of a balanced chromosomal translocation, this technique has the advantage of reaching a diagnosis around the 11th or 12th week of gestation. In experienced hands the procedure-specific risk of miscarriage is up to 1%.
  3. Cordocentesis: Cordocentesis is a technically challenging intervention in which the umbilical vein is entered wherever possible at the site of placental insertion. Cordocentesis can be used for rapid karyotyping or molecular genetic diseases from 16 to 20 weeks depending on indication. Cordocentesis is significant when a rapid result is required late in pregnancy. For example, if there is presence of certain abnormalities detected via ultrasonography or severe growth restriction, which may indicate chromosomal abnormalities. The result of chromosome analysis of lymphocytes from cord blood can be available in three to five days.