Lecture 6: Single nucleotide polymorphisms (SNPs) and Restriction Fragment Length Polymorphisms (RFLPs)

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1 Lecture 6: Single nucleotide polymorphisms (SNPs) and Restriction Fragment Length Polymorphisms (RFLPs) Single nucleotide polymorphisms or SNPs (pronounced "snips") are DNA sequence variations that occur when a single nucleotide (A,T,C, or G) in the genome sequence is altered. For example a SNP might change the DNA sequence AAGGCTAA to ATGGCTAA. For a variation to be considered a SNP, it must occur in at least 1% of the population. SNPs, which make up about 90% of all human genetic variation, occur every 100 to 300 bases along the 3- billion-base human genome. Two of every three SNPs involve the replacement of cytosine (C) with thymine (T). SNPs can occur in both coding (gene) and noncoding regions of the genome. Although more than 99% of human DNA sequences are the same across the population, variations in DNA sequence can have a major impact on how humans respond to disease; environmental insults such as bacteria, viruses, toxins, and chemicals; and drugs and other therapies. SNPs are also evolutionarily stable --not changing much from generation to generation -- making them easier to follow in population studies. SNPs do not cause disease, but they can help determine the likelihood that someone will develop a particular disease. One of the genes associated with Alzheimer's, apolipoprotein E or ApoE, is a good example of how SNPs affect disease development. This gene contains two SNPs that result in three possible alleles for this gene: E2, E3, and E4. Each allele differs by one DNA base, and the protein product of each gene differs by one amino acid. Each individual inherits one maternal copy of ApoE and one paternal copy of ApoE. Research has shown that an individual who inherits at least one E4 allele will have a greater chance of getting Alzheimer's. Apparently, the change of one amino acid in the E4 protein alters its structure and function enough to make disease development more likely. Inheriting the E2 allele, on the other hand, seems to indicate that an individual is less likely to develop Alzheimer's. Of course, SNPs are not absolute indicators of disease development. Someone who has inherited two E4 alleles may never develop Alzheimer's, while another who has inherited two E2 alleles may. ApoE is just one gene that has been linked to Alzheimer's. Like most common chronic disorders such as heart disease, diabetes, or cancer, Alzheimer's is a disease that can be caused by variations in several genes. The polygenic nature of these disorders is what makes genetic testing for them so complicated.

2 I. RFLPs (restriction fragment length polymorphisms) Review of basic techniques: The use of restriction fragment length polymorphisms (RFLPs) and variable length tandem repeats (VNTRs) as genetic markers and tools in DNA fingerprinting is heavily dependent on the use of Southern blotting with appropriate restriction endonucleases and carefully selected probes.

3 Use of RFLPs as genetic markers: When a specific cloned DNA probe is used to analyze a Southern blot of human (or other) DNA, a limited number of restriction fragments of specific and characteristic lengths will be identified. Because single base mutations can either create additional restriction sites or destroy pre-existing sites, DNA preparations from different individuals frequently exhibit different patterns of size distribution of restriction fragments that hybridize with a particular probe. These differences are called restriction fragment length polymorphisms (RFLPs). In many cases, the genetic polymorphisms that generate RFLPs will have no obvious genetic effect because they are located in introns or involve "silent" mutations that convert a codon to different codon specifying the same amino acid. However, they are inherited as codominant Mendelian markers and are extremely useful in studies of human genetic linkage. Annonymous probes: The special advantage of RFLPs as genetic markers is that they do not need to have any special properties other than the existence of the restriction endonuclease that responds to the presence or absence of a particular cut site and the availability of a probe that can be used to visualize the fragments. Any random clone, including sequences located in introns or between genes, that happens to emerge during "shotgun" cloning can potentially be used as a probe. Probes of this sort that do not correspond to

4 any known genes are referred to as anonymous probes. Many useful RFLPs are identified with anonymous probes. Human linkage markers: It is difficult to find suitable linkage markers for human genetic linkage studies. The total number of known genes is still rather small (although it is now growing rapidly because of the human genome project). In addition, many of the genetic loci have been identified only in terms of relatively rare alleles that cause disease phenotypes, with the vast majority of the population carrying the wild-type alleles that do not differ from one individual to another. Codominant expression: RFLP haplotypes (RFLPs carried on single chromosomes in a genome) are stable genetic markers that are inherited in a codominant manner, often with a relatively high frequency of alternative alleles in healthy individuals. This allows them to be used in all types of genetic studies, including analysis of their linkage to the genes responsible for human genetic diseases. Because of their usefulness, large numbers of human RFLPs have been studied in detail, including the chromosomal locations of the DNA sequences responsible for the polymorphisms. Linkage to RFLP haplotypes: Because most human genetic diseases are initially identified only by the disease phenotype, demonstration of linkage to a specific RFLP haplotype is frequently the first step toward identifying the chromosome that carries the disease gene. In addition, a close linkage (identified by a high lod score) can localize the disease gene to a specific region of the chromosome. This in turn provides the starting point for studies leading to the isolation and cloning of the specific gene that is responsible for the disease. Six different examples of identification and cloning of genes responsible for inherited human diseases are presented below, each of which employed a somewhat different experimental approach. Some examples: Neurofibromatosis: Type 1 neurofibromatosis is an autosomal dominant condition associated with a wide range of nervous system defects, including benign tumors and learning disabilities. As described in the textbook (pages ), a search for linkage to specific RFLPs localized the candidate gene to a region near the centromere of human chromosome 17. After the gene was localized as much as possible, chromosome walking was undertaken until a candidate gene was encountered. Its involvement in the disease was verified by sequencing studies that showed mutations in individuals afflicted with the disease. The overall process that led to the discovery of the NF1 gene is called positional cloning. The wild-type gene appears to function in intracellular signal transduction, and more specifically in down-regulating cellular reproduction.

5 Marfan syndrome: A rather different approach was taken to identify the gene that is defective in Marfan syndrome, an autosomal dominant condition that causes alterations in connective tissue. Particular attention was given to genes coding for proteins known to function in various types of connective tissue. A protein known as fibrillin, which is found in tissues affected by Marfan syndrome was identified as a likely candidate. The gene for fibrillin had already been cloned and mapped to the long arm of human chromosome 15. RFLP studies verified a linkage between the inheritance of Marfan syndrome and markers on chromosome 15. Cloning of the fibrillin gene from individuals with Marfan syndrome then verified the substitution of a proline for arginine at position 239 in the protein. The textbook describes this as the candidate gene approach. Huntington disease: The search for the gene responsible for Huntington disease (also known as Huntington's chorea) was described in a previous textbook as an example of the use of RFLPs. Huntington disease is an autosomal dominant degenerative brain disease that usually does not exhibit any obvious symptoms prior to middle age. There is then a progressive loss of motor coordination, accompanied by uncontrolled spontaneous movements, ultimately resulting in death, but only after a prolonged period of increasingly severe symptoms.