Normal Gene variation:
There is normal variation in DNA sequences, this polymorphism occur approximately once in every 500-1000 nucleotides. There are 3, 300, 000 single nucleotide polymorphisms (SNPs) relative to standard initially sequenced human reference genome. Over 80% of SNPs found in DNA have already been found. Each human differs from the next by about 1 bp in every 1, 000 bp and this is what (snips) makes each individual different. There are genomic deletions and insertions of DNA (ie, copy number variations; CNV) as well as single-base substitutions. These are usually occurring in non coding region of DNA. This heritable polymorphism of DNA structure can be associated with certain diseases.
Gene variation causing disease:
Apart from point mutation, disease can be caused by gene variations in any of the steps of central dogma. E.g. β-globin gene located on chromosome 11. The defective production of beta globin gene can lead to various disease and is due to many different lesions in and around the beta-globin gene.
In Fig, LCR is a powerful enhancer. Solid dark boxes represent deletions of that segment leading to various diseases.
In β-thalassemia, various mutation of β-globin gene occurs. Mutation affecting transcription control, nonsense mutation, mutation in RNA processing, and RNA cleavage mutations have been identified. These may be present within Exons or introns.
Point Mutations:
The classic example is sickle cell disease caused by mutation of single base out of 3 X 109 in genome, and T to A DNA substitution resulting in A to U change in mRNA corresponding to the 6th codon of the β-globin gene, this 6th position is changed to Valine in place of Glutamate). This cause structural abnormality of the globin chain. Such point mutations are also observed for β- thalassemia.
Deletions, Insertions, and Rearrangements of DNA:
All these can cause change in gene expression resulting in disease. E.g. β-thalassemia as shown in table above. Deletion of α-globin cluster can cause α-thalassemia. There is strong ethnic association for many of these deletions, E.g. northern Europeans, Filipinos, blacks people have different lesions all resulting in the absence of HbA and α-thalassemia. Deletions of insertions of DNA larger than 50 bp can be detected by Southern blotting and/or PCR.
Prenatal diagnosis of disease:
DNA based technologies has helped in the prenatal diagnosis of genetic abnormalities especially if familial history of disease is present.
Fetal cells obtained from amniotic fluid or from biopsy of the chorionic villi, or white blood cells can be used for karyotyping, which assesses the morphology of metaphase chromosomes. New staining and cell sorting techniques have permitted the rapid identification of trisomies and translocations that produce chromosomes of abnormal lengths.
E.g. In the case of sickle cell disease, the mutation that gives rise to the disease is same as the mutation that gives rise to the polymorphism. Direct detection by RFLP of diseases that result from point mutations is at present limited to only a few genetic diseases.
Initially prenatal diagnosis of HbS was made by analyzing the type of Hb in hemolysate prepared from fetal blood. This invasive procedure led to high mortality rate (about 5%) and diagnosis cannot be carried out until late 2nd trimester when HbS begins to be produced. But now rapid DNA technologies are available to overcome this problem.
E.g. Sickle cell anemia. The substitution of T for A in the template strand of DNA in the β-globin gene changes the sequence in the region that corresponds to the 6th codon and destroys a recognition site for the restriction enzyme MstII (CCTNAGG).
Other MstII sites 5’ and 3’ from this site are not affected and so will not be cut. Incubation of DNA from normal AA, heterozygous (AS), and homozygous (SS) individuals results in 3 different patterns on Southern blot transfer.
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| Fig: RFLP analysis of sickle cell disease in offspring. |
By taking and analyzing amniotic fluid or by chorionic villus biopsy can be analyzed by Southern blot transfer to identify a fetus with abnormality as shown in figure above. Thus this has helped in prenatal diagnosis of disease.
Another example in the prenatal diagnosis of Phenylketonuria (PKU) using RFLP: The gene for phenyl alanine hydroxylase (PAH), the deficient of which leads to PKU, is located on chromosome 12 and contains 13 exons separated by introns. Mutation in this gene do not affect restriction site. For the diagnosis one has to analyze DNA of family members of the afflicted individual to identify markers (RFLP) that are linked to the disease trait. Once these markers are identified, RFLP analysis can be used to carry out prenatal diagnosis. If RFLP is associated with the disease producing gene then by the use of RFLP in family members, it is possible to trace the inheritance of the disease producing DNA within a family without knowledge of nature of genetic defect or its precise location in the genome. The polymorphism may be known from the study of other families with the same disorder. The presence of abnormal PAH genes can be shown using DNA polymorphisms as markers to distinguish between normal and mutant genes.
(Source: Lehninger's Textbook of Biochemistry and Lippincott's Illustrated Biochemistry)
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