Lecture 6: Single nucleotide polymorphisms (SNPs)

Restriction Fragment Length Polymorphisms (RFLPs)

and

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 3billion-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.

I. RFLPs (restriction fragment length polymorphisms)

The use of restriction fragment length
Review of basic techniques:
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.

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

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
464-465), 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.

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.