Read this article to learn about the use of DNA in the diagnosis of infectious and genetic diseases.
A. DNA in the Diagnosis of Infectious Diseases :
The use of DNA analysis (by employing DNA probes) is a novel and revolutionary approach for specifically identifying the disease-causing pathogenic organisms. This is in contrast to the traditional methods of disease diagnosis by detection of enzymes, antibodies etc., besides the microscopic examination of pathogens.
Although at present not in widespread use, DNA analysis may soon take over the traditional diagnostic tests in the years to come. Diagnosis of selected diseases by genetically engineered techniques or DNA probes or direct DNA analysis is briefly described.
Tuberculosis:
Tuberculosis is caused by the bacterium Mycobacterium tuberculosis. The commonly used diagnostic tests for this disease are very slow and sometimes may take several weeks. This is because M. tuberculosis multiplies very slowly (takes about 24 hrs. to double; E. coli takes just 20 minutes to double).
A novel diagnostic test for tuberculosis was developed by genetic engineering, and is illustrated in Fig. 14.3. A gene from firefly, encoding the enzyme luciferase is introduced into the bacteriophage specific for M. tuberculosis. The bacteriophage is a bacterial virus, frequently referred to as luciferase reporter phage or mycophage.
The genetically engineered phage is added to the culture of M. tuberculosis. The phage attaches to the bacterial cell wall, penetrates inside, and inserts its gene (along with luciferase gene) into the M. tuberculosis chromosome. The enzyme luciferase is produced by the bacterium.
When luciferin and ATP are added to the culture medium, luciferase cleaves luciferin. This reaction is accompanied by a flash of light which can be detected by a luminometer. This diagnostic test is quite sensitive for the confirmation of tuberculosis. The flash of light is specific for the identification of M. tuberculosis in the culture. For other bacteria, the genetically engineered phage cannot attach and enter in, hence no flash of light would be detected.
Malaria:
Malaria, mainly caused by Plasmodium falciparum and P. vivax, affects about one-third of the world’s population. The commonly used laboratory tests for the diagnosis of malaria include microscopic examination of blood smears, and detection of antibodies in the circulation. While the former is time consuming and frequently gives false-negative tests, the latter cannot distinguish between the past and present infections.
A specific DNA diagnostic test for identification of the current infection of P. falciparum has been developed. This is carried out by using a DNA probe that can bind and hybridize with a DNA fragment of P.falciparum genome and not with other species of Plasmodium. It is reported that this DNA probe can detect as little as 1mg of P. falciparum in blood or 10 pg of its purified DNA.
Chagas’ Disease:
The protozoan parasite Trypanosoma cruzi causes Chagas’ disease. This disease is characterized by destruction of several tissues (liver, spleen, brain, lymph nodes) by the invading parasite. Chagas’ disease is diagnosed by the microscopic examination of the fresh blood samples. Immunological tests, although available, are not commonly used, since they frequently give false-positive results.
Scientists have identified a DNA fragment with 188-base pair length present in T. cruzi genome. This is however, not found in any other related parasite. A PCR technique is employed to amplify the 188 bp DNA fragment. This can be detected by using polyacrylamide gel electrophoresis. Thus, PCR-based amplification can be effectively used for the diagnosis of Chagas’ disease.
Acquired Immunodeficiency Syndrome (AIDS):
AIDS is caused by the virus, human immunodeficiency virus (HIV). The commonly used laboratory test for detection of AIDS is the detection of HIV antibodies. However, it might take several weeks for the body to respond and produce sufficient HIV antibodies. Consequently, the antibodies test may be negative (i.e., false-negative), although HIV is present in the body. During this period, being a carrier, he/she can transmit HIV to others.
DNA probes, with radioisotope label, for HIV DNA are now available. By using PCR and DNA probes, AIDS can be specifically diagnosed in the laboratory. During the course of infection cycle, HIV exists as a segment of DNA integrated into the T-lymphocytes of the host. The T-lymphocytes of a suspected AIDS patient are isolated and disrupted to release DNA.
The so obtained DNA is amplified by PCR, and to this DNA probes are added. If the HIV DNA is present, it hybridizes with the complementary sequence of the labeled DNA probe which can be detected by its radioactivity. The advantage of DNA probe is that it can detect the virus when there are no detectable antibodies in the circulation.
HIV diagnosis in the newborn:
Detection of antibodies is of no use in the newborn to ascertain whether AIDS has been transmitted from the mother. This is because the antibodies might have come from the mother but not from the virus. This problem can be solved by using DNA probes to detect HIV DNA in the newborn.
Human Papilloma Virus:
Human papilloma virus (HPV) causes genital warts. HPV is also associated with the cervical cancer in women. The DNA probe (trade name Virapap detection kit) that specifically detects HPV has been developed. The tissue samples obtained from woman’s cervix are used. HPV DNA, when present hybridizes with DNA probe by complementary base pairing, and this is the positive test.
Lyme Disease:
Lyme disease is caused by the bacterium, Borrelia burgdorferi. This disease is characterized by fever, skin rash, arthritis and neurological manifestations. The diagnosis of Lyme disease is rather difficult, since it is not possible to see B. burgdorferi under microscope and the antibody detection tests are not very reliable. Some workers have used PCR to amplify the DNA of B. burgdorferi. By using appropriate DNA probes, the bacterium causing Lyme disease can be specifically detected.
Periodontal Disease:
Periodontal disease is characterized by the degenerative infection of gums that may ultimately lead to tooth decay and loss. This disease is caused by certain bacteria. At least three distinct species of bacteria have been identified and DNA probes developed for their detection. Early diagnosis of periodontal disease will help the treatment modalities to prevent the tooth decay.
DNA Probes for Other Diseases:
In principle, almost all the pathogenic organisms can be detected by DNA probes. Several DNA probes (more than 100) have been developed and many more are in the experimental stages. The ultimate aim of the researchers is to have a stock of probes for the detection of various pathogenic organisms—bacteria, viruses, parasites. The other important DNA probes in recent years include for the detection of bacterial infections caused by E. coli (gastroenteritis) Salmonella typhi (food poisoning), Campylobacter hyoitestinalis (gastritis).
Diagnosis of tropical diseases:
Malaria, filariasis, tuberculosis, leprosy, schistosomiasis, leishmaniasis and trypanosomiasis are the tropical diseases affecting millions of people throughout the world. As already described for the diagnosis of malaria caused by P. falciparum, a DNA probe has been developed. A novel diagnostic test, by genetic manipulations, has been devised for the diagnosis of tuberculosis. Scientists are continuously working to develop better diagnostic techniques for other tropical diseases.
B. DNA in the Diagnosis of Genetic Diseases:
Traditional laboratory tests for the diagnosis of genetic diseases are mostly based on the estimation of metabolites and/or enzymes. This is usually done after the onset of symptoms. The laboratory tests based on DNA analysis can specifically diagnose the inherited diseases at the genetic level.
DNA-based tests are useful to discover, well in advance, whether the individuals or their offspring’s are at risk for any genetic disease; Further, such tests can also be employed for the prenatal diagnosis of hereditary disorders, besides identifying the carriers of genetic diseases.
By knowing the genetic basis of the diseases, the individuals can be advised on how to limit the transmission of the disease to their offspring’s. It may also be possible, in due course of time, to treat genetic diseases by appropriate gene therapies. Theoretically, it is possible to develop screening tests for all single-gene diseases. Some of the important genetic diseases for which DNA analysis is used for diagnosis are briefly described.
Cystic Fibrosis:
Cystic fibrosis (CF) is a common and fatal hereditary disease. The patients produce thick and sticky mucus that clogs lungs and respiratory tract. Cystic fibrosis is due to a defect in cftr gene that encodes cystic fibrosis trans membrane regulator protein, cftr gene is located on chromosome 7 in humans, and a DNA probe has been developed to identify this gene.
The genetic disease cystic fibrosis is inherited by a recessive pattern, i.e., the disease develops when two recessive genes are present. It is now possible to detect CF genes in duplicate in the fetal cells obtained from samples of amniotic fluid. As the test can be done months before birth, it is possible to know whether the offspring will be a victim of CF. One group of researchers have reported that CF gene can be detected in the eight-celled embryo obtained through in vitro fertilization.
Sickle-Cell Anemia:
Sickle-cell anemia is a genetic disease characterized by the irregular sickle (crescent like) shape of the erythrocytes. Biochemically, this disease results in severe anemia and progressive damage to major organs in the body (heart, brain, lungs, and joints).
Sickle-cell anemia occurs due to a single amino acid change in the β-chain of hemoglobin. Specifically, the amino acid glutamate at the 6th position of β-chain is replaced by valine. At the molecular level, sickle-cell anemia is due to a single-nucleotide change (A → T) in the β-globin gene of coding (or antisense) strand.
In the normal β-globin gene the DNA sequence is CCTGAGGAG, while in sickle-cell anemia, the sequence is CCTGTGGAG. This single-base mutation can be detected by using restriction enzyme Mstll to cut DNA fragments in and around β-globin gene, followed by the electrophoretic pattern of the DNA fragments formed.
The change in the base from A to T in the β-globin gene destroys the recognition site (CCTGAGG) for Mstll (Fig. 14.4). Consequently, the DNA fragments formed from a sickle-cell anemia patient for β-globin gene differ from that of a normal person. Thus, sickle-cell anemia can be detected by digesting mutant and normal β-globin gene by restriction enzyme and performing a hybridization with a cloned β-globin DNA probe.
Single-nucleotide polymorphisms:
The single base changes that occur in some of the genetic diseases (e.g., sickle-cell anemia) are collectively referred to as single-nucleotide polymorphisms (SNPs, pronounced snips). It is estimated that the frequency of SNPs is about one in every 1000 bases. Sometimes SNPs are associated with amino acid change in the protein that is encoded. A point mutation in α1-antitrypsin gene is also a good example of SNPs, besides sickle-cell anemia.
Duchenne’s Muscular Dystrophy:
Duchenne’s muscular dystrophy (DMD) is a genetic abnormality characterized by progressive wasting of leg and pelvic muscles. It is a sex-linked recessive disease that appears between 3 and 5 years of age. The affected children are unsteady on their feet as they lose the strength and control of their muscles. By the age of ten, the victims of DMD are confined to wheel chair and often die before reaching 20 years age.
The patients of DMD lack the muscle protein, namely dystrophin which gives strength to the muscles. Thus, DMD is due to the absence of a gene encoding dystrophin. For specific diagnosis of Duchenne’s muscular dystrophy, a DNA probe to identify a segment of DNA that lies close to defective gene (for dystrophin) is used. This DNA segment, referred to as restriction fragment length polymorphism (RFLP), serves as a marker and can detect DMD with 95% certainty.
In the DNA diagnostic test using RFLP for DMD, DNA samples must be obtained from as many blood relatives (parents, grand-parents, uncles, aunts etc.) as possible. The RFLP patterns, constructed for the entire family are thoroughly checked for the affected and unaffected relatives. This is required since there is a wide variation in RFLPs from family to family. Thus, there is no single identifying test for the diagnosis of genetic diseases based on RFLPs analysis.
Huntington’s Disease:
Huntington’s disease is a genetic disease (caused by a dominant gene) characterized by progressive deterioration of the nervous system, particularly the destruction of brain cells. The victims of this disease (usually above 50 years of age) exhibit thrashing (jerky) movements and then insanity [older name was Huntington’s chorea; chorea (Creek) means to dance]. Huntington’s disease is invariably fatal.
The molecular basis of Huntington’s disease has been identified. The gene responsible for this disease lies on chromosome number 4, and is characterized by excessive repetition of the base triplet CAG. The victims of Huntington’s disease have CAG triplet repeated 42-66 times, against the normal 11-34 times.
The triplet CAG encodes for the amino acid glutamine. It is believed that the abnormal protein (with very high content of glutamine) causes the death of cells in the basal ganglia (the part of the brain responsible for motor function).
Huntington’s disease can be detected by the analysis of RFLPs in blood related individuals. The clinical manifestations of this disease are observed after middle age, and by then the person might have already passed on the defective gene to his/ her offspring’s.
Fragile X Syndrome:
Fragile X syndrome, as the name indicates, is due to a genetic defect in X chromosome (a sex chromosome) and affects both males and females. The victims of this disease are characterized by mental retardation. Researchers have found that sufferers of fragile X syndrome have the three nucleotide bases (CGG) repeated again and again.
It is believed that these tri-nucleotide repeats block the transcription process resulting in a protein deficiency. This protein is involved in the normal function of the nerve cells, and its deficiency results in mental retardation. A DNA probe has been developed for the detection of fragile X syndrome in the laboratory.
Other Triple Repeat Diseases:
Excessive repetition of triplet bases in DNA are now known to result in several diseases which are collectively referred to as triple repeat diseases. Besides Huntington’s disease and fragile X syndrome, some more triple repeats are given below.
Friedreich’s ataxia:
The tri-nucleotide GAA repeats 200 to 900 times on chromosome 9 in Friedreich’s ataxia. This disease is associated with degradation of spinal cord. Spinocerebellar ataxia is another triplet disease, characterized by neuromuscular disorder, and is due to tri-nucleotide repeats of CAG by 40 to 80 times on chromosome 6.
There are a few triple repeat diseases in which the repeats tend to increase with each generation and the diseases become more severe. This also results in the onset of clinical manifestations at early ages. Kennedy’s disease, also called spinobulbar muscular atropy (CAG repeat) and myotonic dystrophy (CTG) are good examples.
Are triple repeat diseases confined to humans?
Triple repeat diseases have so far not been detected in any other organisms (bacteria, fruit flies, other mammals) except in humans. More studies however, may be needed to confirm this. The occurrence of triple repeat diseases indicates that the structure of DNA may be rather unstable and dynamic. This is in contrast to what molecular biologists have been thinking all along.
Alzheimer’s Disease:
Alzheimer’s disease is characterized by loss of memory and impaired intellectual function (dementia). The victims of this disease cannot properly attend to their basic needs, besides being unable to speak and walk. The patients of Alzheimer’s disease were found to have a specific protein, namely amyloid in the plaques (or clumps) of dead nerve fibers in their brains. A group of researchers have identified a specific gene on chromosome 21 that is believed to be responsible for familial Alzheimer’s disease.
A DNA probe has been developed to locate the genetic marker for Alzheimer’s disease. The present belief is that many environmental factors and a virus may also be responsible for the development of this disease. It may be possible that in the individuals with genetic predisposition, the outside factors may be stimulatory for the onset of the disease.
Amyotrophic Lateral Sclerosis:
Amyotrophic lateral sclerosis (ALS) is characterized by degenerative changes in the motor neurons of brain and spinal cord. A gene to explain the inherited pattern of ALS was discovered. The gene, known as sodl, encoding for the enzyme superoxide dismutase is located on chromosome 21. This gene was found to be defective in families suffering from amyotrophic lateral sclerosis. In fact, certain point mutations in the sodl resulting in single amino acid changes in superoxide dismutase have been identified.
Superoxide dismutase is a key enzyme in eliminating the highly toxic free radicals that damage the cells (free radicals have been implicated in aging and several disease e.g. cancer, cataract, Parkinson’s disease, Alzheimer’s disease). On the basis of the function of superoxide dismutase, it is presumed that ALS occurs as a result of free radical accumulation due to a defective enzyme (as a consequence of mutated gene sodl). The deleterious effects of free radicals can be reduced by administering certain compounds such as vitamins C and E.
Another group of workers have reported that the defective superoxide dismutase cannot control a transporter protein responsible for the removal of the amino acid glutamate from the nerve cells. As a result, large quantities of glutamate accumulate in the nervous tissue leading to degenerative changes.
Cancers:
It is now agreed that there is some degree of genetic predisposition for the occurrence of cancers, although the influence of environmental factors cannot be underestimated. In fact, cancer susceptible genes have been identified in some families e.g., genes for melanoma susceptibility in humans are located on chromosomes 1 and 9.
p53 Gene:
The gene p53 encodes for a protein with a molecular weight 53 kilo Daltons (hence the name). It is believed that the protein produced by this gene helps DNA repair and suppresses cancer development. Certain damages that occur in DNA may lead to unlimited replication and uncontrolled multiplication of cells.
In such a situation, the protein encoded by p53 gene binds to DNA and blocks replication. Further, it facilitates the faulty DNA to get repaired. The result is that the cancerous cells are not allowed to establish and multiply. Thus, p53 is a cancer-suppressor gene and acts as a guardian of cellular DNA.
Any mutation in the gene p53 is likely to alter its tumor suppressor function that lead to cancer development. And in fact, the altered forms of p53 recovered from the various tumor cells (breast, bone, brain, colon, bladder, skin, lung) confirm the protective function of p53 gene against cancers.
It is believed that the environmental factors may cause mutations in p53 gene which may ultimately lead to cancer. Some of the mutations of p53 gene may be inherited, which probably explains the occurrence of certain cancers in some families.
Genes of breast cancer:
Two genes, namely BRCAI and BRCAII, implicated in certain hereditary forms of breast cancer in women, have been identified. It is estimated that about 80% of inherited breast cancers are due to mutations in either one of these two genes — BRCAI or BRCAII. In addition, there is a high risk for ovarian cancer due to mutations in BRCAI.
It is suggested that the normal genes BRCAI and BRCAII encode proteins (with 1863 and 3418 amino acids respectively) that function in a manner comparable to gene p53 protein (as described above). As such, BRCAI and BRCAII are DNA- repair and tumor-suppressor genes. Some researchers believe that these two proteins act as gene regulators. Diagnostic tests for the analysis of the genes BRCAI and BRCAII were developed. Unfortunately, their utility is very limited, since there could be hundreds of variations in the base sequence of these genes.
Genes of colon cancer:
The occurrence of colon cancer appears to be genetically linked since it runs in some families. Some researchers have identified a gene linked with hereditary non-polyposis colon cancer or HNPCC (sometimes called Lynch syndrome). This gene encoded a protein that acts as a guardian and brings about DNA repair whenever there is a damage to it.
However, as and when there is a mutation to this protective gene, an altered protein is produced which cannot undo the damage done to DNA. This leads to HNPCC. It is estimated that the occurrence of this altered gene is one in every 200 people in general population.
Microsatellite marker genes:
Microsatellites refer to the short repetitive sequences of DNA that can be employed as markers for the identification of certain genes. For colon cancer, microsatellite marker genes have been identified on chromosome 2 in humans. There is a lot of variability in the sequence of microsatellites.
Early detection of the risk for colon cancer by DNA analysis is a boon for the would be victims of this disease. The suspected individuals can be periodically monitored for the signs and treated appropriately. Unlike many other cancers, the chances of cure for colon cancer are reasonably good.
Gene of retinoblastoma:
Retinoblastoma is a rare cancer of the eye. If detected early, it can be cured by radiation therapy and laser surgery or else the eyeball has to be removed. Scientists have identified a missing or a defective (mutated) gene on chromosome number 13, being responsible for retinoblastoma. The normal gene when present on chromosome 13 is anticancer and does not allow retinoblastoma to develop.
Diabetes:
Diabetes mellitus is a clinical condition characterized by increased blood glucose level (hyperglycemia) due to insufficient or inefficient (incompetent) insulin. In other words, individuals with diabetes cannot utilize glucose properly in their body.
A rare form of type II diabetes (i.e., non-insulin dependent diabetes mellitus, NIDDM) is maturity onset diabetes of the young (MODY). MODY, occurring in adolescents and teenagers is found to have a genetic basis. A gene, synthesizing the enzyme glucokinase, located on chromosome 7, is found to be defective in MODY patients.
Glucokinase is a key enzyme in glucose metabolism. Besides its involvement in the metabolism, glucokinase in the pancreatic cells serves as a detector for glucose concentration in the blood. This detection stimulates β-cells of the pancreas to secrete insulin. A gene modification that results in a defective or an altered glucokinase hampers pancreatic insulin secretion. Later work has shown that glucokinase gene is defective in the common form of type II diabetes.
DNA probes for type II diabetes:
The glucokinase genes from normal and type II diabetes patients were cloned and scanned with DNA probes. It was found that a single base mutation of the gene led to a defective glucokinase production that is largely responsible for MODY, and also a majority of individuals with type II diabetes. Later, some workers reported a possibility of at least a dozen mutations in glucokinase gene for type II diabetes.
Genes responsible for type I diabetes:
Type I diabetes or insulin-dependent diabetes mellitus (IDDM) mainly occurs in childhood, particularly between 12-15 years of age. IDDM is characterized by almost total deficiency of insulin. Researchers have identified at least 18 different chromosome regions linked with type I diabetes. These DNA sequences are located on chromosomes 6, 11 and 18.
Obesity:
Obesity is an abnormal increase in the body weight due to fat deposition. Men and women are considered obese if their weight due to fat, respectively exceeds more than 20% and 25% of the body weight. Obesity increases the risk of high blood pressure, diabetes, atherosclerosis and other life-threatening conditions.
Although many believed that obesity could be genetically inherited, the molecular basis was not known for long. It was in 1994, a group of workers identified a mutated gene that caused obesity in mice. Later, a similar gene was found in humans also.
The gene designated ob (for obese) is located on chromosome 6 in mouse. The DNA of ob gene contains 650 kb and encodes a protein with 167 amino acids in adipose tissue. This protein is responsible to keep the weight of the animals under control.
The genetically obese mice have mutated ob gene and therefore the weight-control protein is not produced. It is believed that this protein functions like a hormone, acts on the hypothalamus, and controls the site of hunger and energy metabolism (these two factors are intimately linked with obesity).
With the discovery of ob gene, the treatment for inherited obesity may soon become a reality. In fact, one multinational biotechnology company has started producing ob protein that can be used for weight reduction in experimental mice. Besides the ob gene, a few other genes (fat gene, tub gene) that might be associated with obesity have also been discovered.
DNA Analysis for Other Human Diseases:
There is a continuous search for the identification of more and more genes that are responsible for human diseases. Such an approach will ultimately help in the specific diagnosis of these diseases before their actual occurrence. In addition to human diseases described above, some more are given below.
Deafness:
The deafness, inherited in some families, has genetic basis. A team of workers have identified a gene on chromosome 5, encoding a protein that facilitates the assembly of actin (protein) molecules in the cochlea of inner ear. The association of actin is very essential for the detection of sound waves by the ear.
A mutation of the gene on chromosome 5 results in a defective protein synthesis and non- assembly of actin molecules which cause deafness. Some other genes, besides the one described here, have also been found to be associated with deafness.
Glaucoma:
Glaucoma is a disease of the eye that may often lead to blindness. It occurs as a result of damage to the optic nerve due to pressure that builds up in the eye. A gene responsible for the hereditary glaucoma in teenagers has been detected on chromosome 1. Another group of researchers have found a gene on chromosome 3 which is linked with the adult-onset glaucoma.
Baldness:
There is an inherited form of baldness, called alopecia universals. This is found to be associated with a gene located on chromosome 12.
Parkinson’s disease:
Parkinson’s disease is a common disorder in many elderly people, with about 1% of the population above 60 years being affected. It is characterized by muscular rigidity, tremors, expressionless face, lethargy, involuntary movements etc. In the victims of Parkinson’s disease, there is degeneration of brain cells, besides a low concentration of dopamine (a neurotransmitter).
Researchers have identified that a gene-encoded protein namely α-synuclein plays a significant role in the development of Parkinson’s disease. An altered form of α-synuclein (due to a mutation in the gene) accumulates in the brain as Lewy bodies. This is responsible for nerve cells degeneration and their death in the Parkinson’s disease.
Hemochromatosis:
Hemochromatosis is an iron-overload disease in which iron is directly deposited in the tissues (liver, spleen, heart, pancreas and skin). An abnormal gene on chromosome 6 is linked with hemochromatosis. The amino acid tyrosine, in the normal protein encoded by this gene is replaced by cysteine. This abnormal protein is responsible for excessive iron absorption from the intestine which accumulates in the various tissues leading to their damage and malfunction.
Menke’s disease:
Menke’s disease, a copper deficiency disorder, is characterized by decreased copper in plasma, depigmentation of hair, degeneration of nerve cells and mental retardation. A gene located on X-chromosome, encoding a transport protein, is linked with Menke’s disease. A defect in the gene, consequently in the protein, impairs copper absorption from the intestine.
Gene Banks—A Novel Concept:
As the search continues by scientists for the identification of more and more genes responsible for various diseases, the enlightened public (particularly in the developed countries), is very keen to enjoy the fruits of this research outcome. As of now, DNA probes are available for the detection a limited number of diseases. Researchers continue to develop DNA probes for a large number of genetically predisposed disorders.
Gene banks are the centres for the storage of individual’s DMAs for future use to diagnose diseases. For this purpose, the DNA isolated from a person’s cells (usually white blood cells) is stored. As and when a DNA probe for the detection of a specific disease is available, the stored DNA can be used for the diagnosis or risk assessment of the said genetic disease.
In fact, some institutions have established gene banks. They store the DNA samples of the interested customers at a fee (one firm was charging $ 200) for a specified period (say around 20-25 years). For the risk assessment of any disease, it is advisable to have the DNAs from close relatives of at least 2-3 generations.