DNA Polymorphism...
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THE NATIONAL MEDICAL JOURNAL OF INDIA VOL,2, NO.3 109 DNA Polymorphism in Clinical Medicine For more than a decade recombinant DNA technology has contributed to a better understanding of disease mechanisms, provided new means of diagnosis and has opened the exciting possibility of gene therapy. While its greatest impact thus far has been in genetic disorders, recent technological advances permit the application of recombinant DNA methods to the diagnosis of micro- bial and parasitic diseases as well as viral infections. The term restriction fragment length polymorphism (RFLP) describes the variation in the DNA fragment length generated either through gross gene abnormalities (such as deletions, insertions or rearrangements) or point muta- tions in critical regions of genes. So far more than 1200 polymorphic markers have been identified in the human genome. These can be defined by digesting DNA from normal and affected persons with restriction endonucleases (bacterial enzymes that recognize and cleave at sequence specific sitesin DNA), resolv- ing the resulting DNA fragments by agarose gel electrophoresis, immobilizing the DNA on a solid support and hybridizing with gene specific DNA probes. When a gene responsible for a disease is cloned and characterized, several polymorphisms are usually identified either within the gene or very close to it. The DNA polymorphism, while not always responsible for the genetic disorder, serves to mark the normal and abnormal alleles on the two different chromo- somes of an individual. RFLP analysis is a very powerful tool because even those diseases can be studied whose molecular defects are unknown such as Huntington's chorea and cystic fibrosis. Over 70 genes responsible for genetic disorders have been cloned and more will be detected by DNA polymorphic markers." RFLP linkage analysis is currently being applied for carrier detection and prenatal diagnosis of disorders such as cystic fibrosis, Huntington's chorea, Duchenne muscular dystrophy, haemophilia A, familial hypercholesterolaemia, the Lesch-Nyhan syndrome, the thalassaemias and Tay-Sachs disease. Haemoglobinopathies and thalassaemias are disorders that have been most extensively studied at the DNA level. The stepwise refinement of DNA-based detection methods, as applied in the diagnosis of sickle-cell anaemia, is detailed below. The first ever DNA polymorphic site detected involved an Hpa I recogni- tion site located 5 kilobase (kb) downstream of the B-globin gene. Kan and Dozy found a 13 kb RFLP that had a high frequency of association with the ~-globin sickle cell (~.S) while the normal allele (~A) gave a 7.6 kb Hpa I fragment. 2 It was observed that 87% of the American population's sickle cell genes were associated with the 13 kb RFLP while an association of only 3% was found for the normal ~-globin allele. In the second stage of diagnostic technology development, DNA sequence information of the ~s mutation permitted the evolution of more precise diagnostic methods. Initially the restriction enzyme Dde I and subsequently Mst II was used to detect the ~.smutant sequence. Since ~A and [jsMst II fragments were associated with 1.15 kb and 1.35 kb respec- tively, a precise diagnosis by DNA hybridization analysis became possible. However, only a limited number of disease-associated inheritable mutations can be assessed by this technique because it depends on a site-specific mutation altering a restriction endonuclease recognition site. For instance, a second mutant allele ~c did not alter the endonuclease recognition site of either Dde I or Mst II. Synthetic oligodeoxynucleotide probes marked the next technical advance since they did not depend on restriction enzyme recognition sites for the unambiguous detection of point mutations. The rationale of this approach is that hybrids between synthetic oligonucleotides that form a perfect match with genomic sequences are more stable than those that exhibit a single mismatch. Using separate allele-specific oligodeoxynucleotides (ASO) that were 19 nucleotides long (19-mer) and perfectly matched the point mutation and flank- ing sequences, procedures were evolved for the detection of [jA, ~s and ~c
Transcript of DNA Polymorphism...
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THE NATIONAL MEDICAL JOURNAL OF INDIA VOL,2, NO.3 109
DNA Polymorphism in Clinical MedicineFor more than a decade recombinant DNA technology has contributed to abetter understanding of disease mechanisms, provided new means of diagnosisand has opened the exciting possibility of gene therapy. While its greatestimpact thus far has been in genetic disorders, recent technological advancespermit the application of recombinant DNA methods to the diagnosis of micro-bial and parasitic diseases as well as viral infections.
The term restriction fragment length polymorphism (RFLP) describes thevariation in the DNA fragment length generated either through gross geneabnormalities (such as deletions, insertions or rearrangements) or point muta-tions in critical regions of genes. So far more than 1200 polymorphic markershave been identified in the human genome. These can be defined by digestingDNA from normal and affected persons with restriction endonucleases (bacterialenzymes that recognize and cleave at sequence specific sitesin DNA), resolv-ing the resulting DNA fragments by agarose gel electrophoresis, immobilizingthe DNA on a solid support and hybridizing with gene specific DNA probes.When a gene responsible for a disease is cloned and characterized, severalpolymorphisms are usually identified either within the gene or very close to it.The DNA polymorphism, while not always responsible for the genetic disorder,serves to mark the normal and abnormal alleles on the two different chromo-somes of an individual. RFLP analysis is a very powerful tool because eventhose diseases can be studied whose molecular defects are unknown such asHuntington's chorea and cystic fibrosis. Over 70 genes responsible for geneticdisorders have been cloned and more will be detected by DNA polymorphicmarkers." RFLP linkage analysis is currently being applied for carrier detectionand prenatal diagnosis of disorders such as cystic fibrosis, Huntington's chorea,Duchenne muscular dystrophy, haemophilia A, familial hypercholesterolaemia,the Lesch-Nyhan syndrome, the thalassaemias and Tay-Sachs disease.
Haemoglobinopathies and thalassaemias are disorders that have been mostextensively studied at the DNA level. The stepwise refinement of DNA-baseddetection methods, as applied in the diagnosis of sickle-cell anaemia, is detailedbelow. The first ever DNA polymorphic site detected involved an Hpa I recogni-tion site located 5 kilobase (kb) downstream of the B-globin gene. Kan andDozy found a 13 kb RFLP that had a high frequency of association with the~-globin sickle cell (~.S)while the normal allele (~A) gave a 7.6 kb Hpa I fragment. 2It was observed that 87% of the American population's sickle cell genes wereassociated with the 13 kb RFLP while an association of only 3% was found forthe normal ~-globin allele. In the second stage of diagnostic technologydevelopment, DNA sequence information of the ~s mutation permitted theevolution of more precise diagnostic methods. Initially the restriction enzymeDde I and subsequently Mst II was used to detect the ~.smutant sequence. Since~A and [jsMst II fragments were associated with 1.15 kb and 1.35 kb respec-tively, a precise diagnosis by DNA hybridization analysis became possible.However, only a limited number of disease-associated inheritable mutationscan be assessed by this technique because it depends on a site-specific mutationaltering a restriction endonuclease recognition site. For instance, a second mutantallele ~c did not alter the endonuclease recognition site of either Dde I or MstII. Synthetic oligodeoxynucleotide probes marked the next technical advancesince they did not depend on restriction enzyme recognition sites for theunambiguous detection of point mutations. The rationale of this approach isthat hybrids between synthetic oligonucleotides that form a perfect match withgenomic sequences are more stable than those that exhibit a single mismatch.Using separate allele-specific oligodeoxynucleotides (ASO) that were 19nucleotides long (19-mer) and perfectly matched the point mutation and flank-ing sequences, procedures were evolved for the detection of [jA, ~s and ~c
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alleles.' However, in general, the technique lacks the desirable level of specificityand requires DNA derived from at least 106 human cells. This problem hasbeen brilliantly circumvented by the development of the polymerase chainreaction (PCR) for the in vitro amplification of the defective gene using sequence-specific primers. 4 The sensitivity of the ASO method has been improved by theamplification of ~-globin DNA sequences in the target DNA using the PCR.sIn the West, ASO-based diagnosis of adult carriers and affected newborns isbeing carried out for sickle cell anaemia, phenylketonuria and a-I-antitrypsindeficiency.
In addition to genetic disorders, RFLP patterns have also been used incancer diagnosis. Certain activating point mutations in the ras gene generateRFLPs which have been used to identify the presence of transforming ras genesin human tumours such as those of the lung," urinary tract? and thehaematopoietic system.f Oligodeoxynucleotide probes have also been used todetect mutations but this methodology is limited by the number of differentspecific probes that would be required to cover the gamut of possible substitu-tions in the ras gene.?
RFLP analysis is an important tool in the study of the epidemiology of disease-causing agents as well as in the study of pathogenic mechanisms in bacteria.However, species-specific DNA probes will find more practical use in thediagnosis of infectious diseases caused by parasites, bacteria and viruses.
RFLP analysis has found widespread application in research and clinicalmedicine in developed countries. However, this mode of disease diagnosis isnot being employed on a regular basis in any hospital in India. In this countrydiseases such as sickle cell anaemia, the {3-thalassaemias,haemoglobin E, glucose-6-phosphate dehydrogenase deficiency, Turner's syndrome, Klinefelter'ssyndrome, Down's syndrome, etc. are of clinical importance and can bestudied. In addition, there is a need to study neural tube defects and orofacialclefts which are multifactorial polygenic birth detects.P-"
Screening of affected families with cloned human DNA probes for linkagedisequilibrium will pave the way for genetic counselling for these disorders.The identification of polymorphic DNA markers linked to diseases that areprevalent in India constitutes the first step in the management of genetic disorders.This could be followed by the establishment of regional clinics and laboratoriesfor patient examination and sample collection.
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gene: Relationship to sickle mutation. Proc Nail Acad Sci USA 1978;75:5631-5.3 Conner BJ, Reyes AA, Morin C, Itakura K, Teplitz RL, Wallace RB. Detection of sickle cell
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5 Saiki RK, Bugawan TL, Horn GT, Mullis KB, Erlich HA. Analysis of enzymatically amplifiedbeta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes. Nature1986;324:163-6.
6 Santos E, Martin-Zanca D, Reddy EP, Pierotti MA, Della Porta G, Barbacid M. Malignantactivation of a K-rasoncogene in lung carcinoma but not in normal tissue of the same patient.Science 1984;223:661-4.
7 Fujita J, Yoshida 0, Yuasa Y, Rhim JS, Hatanaka M, Aaronson SA. Ha-ras oncogenes areactivated by somatic alterations in human urinary tract tumours. Nature 1984;309:464-6.
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Approaches to Health Care. Technical Report 11. Delhi:NIHFW, 1986:97-103.11 Verma l'C. High frequency of neural tube defects in North India. Lancet 1978;1:879-80.
