|
 DNA
is a biological polymer that encodes information essential for all physiological
processes. The cell devotes considerable resources to maintain the integrity
of this information. High priority is given to repairing DNA damage in
an ongoing manner, and stringent proofreading mechanisms are in place
in dividing cells. Despite these safeguards, however, alterations do occur
to the original sequence. These changes may have no effect or can actually
benefit the organism as occurs during evolutionary changes. When the alterations
are deleterious we recognize these changes as mutations.
Mutations
may cause inherited disease when they are present in parental gametes
and are passed on to the next generation. They may cause acquired disease
when they develop de novo in somatic cells. An example of the former would
include achondroplasia, a disorder in which affected individuals have
short-limbed dwarfism. This illness results from a mutation in the fibroblast
growth factor receptor-3 gene (FGFR3). Examples of acquired disorders
are characteristically found in neoplasms, such as those affecting the
p53 tumor suppressor gene. A key distinction between these 2 forms of
mutations is that inherited mutations are present in all cells of the
offspring, whereas acquired mutations affect only a subgroup of cells
in an individual.
Ten
percent of the DNA in the human genome codes for genes. This indicates
that a random mutation is more likely to affect intergenic DNA than DNA
coding for proteins. While the principal disease-causing effect of mutations
seems to be on coding sequences within genes, it is now appreciated that
some mutations of the DNA lying between genes have functional consequences
on gene expression. This realization is based on the recognition that
the intergenic DNA contains sequences essential for the normal regulation
of nearby genes. This regulation is usually referred to as epigenetic
(epi: upon, higher than), so mutations are also classified as genetic
(affecting coding sequences) or epigenetic (affecting gene regulation).
Most
genes may be divided into functional regions. The promoter area is where
the transcriptional machinery gains access to the DNA strand to initiate
transcription. Nearby regulatory sequences determine the correct timing
of transcription, the level of gene expression, and the specific cell
type in which transcription will occur. In addition to these regulatory
regions, a gene also contains the exons which actually encode for protein
and which must be spliced together to form the processed or mature messenger
RNA (mRNA) molecule. The mRNA is then read by the translational machinery
as a sequence of triplets. Each triplet of nucleotides encodes individual
amino acids that are strung together to form a protein molecule. The sequence
of triplets is termed the coding sequence or open reading frame of the
mRNA. Much of this material may be reviewed in 2 earlier columns on transcriptional
control (April and May, 1998).
When
the DNA of a gene is damaged, the cell attempts to repair it. For the
most part, these repairs are successful and the cell resumes its normal
activity. Occasionally, the repairs are not successful and mutations arise.
If the mutation involves the coding region DNA, it may or may not have
an effect on the function of the protein. For example, one of the DNA
triplets encoding the amino acid leucine is GAA. DNA damage may change
this to GAC. The latter triplet also encodes leucine, so no alteration
in amino acid sequence of the protein results. If, however, the change
were from GAA to GCA, the amino acid encoded is now arginine. This change
might still not affect the activity of the protein. If the leucine serves
a critical role for the protein, then its replacement with an arginine
would in all likelihood alter its function. This type of mutation is recognized
as a missense mutation.
The
integrity of the process allowing translation of mRNA to protein depends
on the maintenance of the series of triplets in an unbroken manner. When
a single nucleotide has been changed, the mutation is termed a point mutation.
When several nucleotides are either removed or added, the mutation is
termed a deletion or an insertion. If the deletion or insertion occurs
to a number of nucleotides other than 3 (or a multiple thereof) in the
coding sequence, then the sense of the coding sequence message is lost
downstream of the mutation. For example, if a series of triplet nucleotides
is represented by the series of numbers 123 123 123 123 123 . . . , the
insertion of an additional 2 nucleotides would cause the following pattern:
123 1NN 231 231 231 231 . . . . This usually gives rise to the premature
termination of the protein as one of the codons that now lies beyond the
mutation will have been changed into a termination signal. Moreover, the
protein that is produced will bear no resemblance to the original protein
beyond the point of the mutation. The end result of such mutations are
often nonfunctional proteins. Deletions and insertions that cause alterations
in the frame of reference for the amino acid-encoding triplets are referred
to as nonsense mutations.
On
occasion, the mutation that is present affects the processing of the RNA
molecule. This occurs when the mutation lies within regulatory sequences.
For example, the formation of exons depends on specific nucleotide sequences
flanking the exons and these sequences must be present for normal RNA
processing to occur. Mutations affecting these splice donor and acceptor
sites can cause exons to be lost or introns to be included in the processed
mRNA. These insertions and deletions can also affect the function of the
proteins produced.
RNA
is not merely a passive molecular messenger. The stability of RNA is variable,
and this stability is affected by the nucleotide sequence that is present.
Evolutionary selection dictates that functional mRNA molecules encoded
by the genomes of complex organisms are stable enough to allow translation
within the cell. Thus, a further mechanism that leads to a failure of
protein formation appears to be the generation of unstable mRNAs by mutation
of stabilizing nucleotide sequences.
Mutations
do not always confer a loss of function. Some mutations alter the protein
to render it continuously active by removing the normal physiological
mechanisms that normally modulate its activity. Such a mechanism accounts
for the dominant inheritance pattern of certain genetic diseases. In these
disorders, only 1 of the 2 alleles at a particular locus is mutated while
the other allele produces a normal protein. However, the normal copy is
unable to compensate for the overactive mutated form.
An
example of such a process is multiple endocrine neoplasia, type IIA (MEN2A).
The mutations causing this disorder affect the RET proto-oncogene
on 10q11.2. This protein normally resides on the plasma membrane and serves
as a receptor for a specific growth factor. Upon binding of the proper
ligand, a signal is transferred to the interior of the cell and a specific
pathway is activated. The mutation causes an alteration of cysteine residues
in the molecule's extracellular domain. This induces the dimerization
of the RET receptor on the cell surface and leads to the unregulated and
continuous activation of the signaling pathway inside the cell. Even the
presence of normal copies of the receptor is unable to modify the effects
of the overactive form. This type of mutation is referred to as a gain
of function mutation.
RET
is a gene that also exhibits the interesting phenomenon of pleiotropy.
Pleiotropy is the ability of different mutations within the same gene
to cause distinct phenotypes. As mentioned, a gain of function mutations
of RET leads to MEN2A. Loss of function mutations, on the other
hand, leads to a phenotype in which the normal nervous supply fails to
form in the colon, leading to a disorder termed Hirschsprung disease.
No endocrine tumors occur in Hirschsprung disease, and colonic aganglionosis
is very rare in MEN2A. The mutations that occur in Hirschsprung disease
are once again located in the RET gene, but they do not cause overactivity
of the receptor. Instead, they arise in different parts of the receptor
molecule and result in its inability to function as a receptor at all.
These mutations either disturb its ability to bind to the ligand or interfere
with the intracellular transmission of the signal.
The
methods by which mutations are detected are rapidly increasing. Direct
sequencing of coding regions is the most direct way of finding a mutation,
but the presence of multiple exons and large coding sequences can make
this process costly and time-consuming. Indirect means of determining
the presence of a mutation include the comparison of electrophoretic mobility
of single stranded DNA isolated from patients and from normal individuals.
Alterations in the nucleotide sequence will cause mobility changes and
will target the DNA for subsequent sequencing experiments. Such techniques
are particularly useful for assessing large genes with numerous exons
where mutations causing disease can occur at multiple loci.
So
far, the focus has been on subtle mutations affecting at most several
base pairs at a time. Larger-scale events also occur. A germline mutation
such as trisomy 21, where an entire extra chromosome 21 is inherited by
the offspring, causes the Down syndrome phenotype. Monosomy for the X
chromosome (only one X chromosome, without a second sex chromosome, X
or Y) can result in spontaneous abortion or, less frequently, in girls
with the Turner syndrome phenotype.
Deletions
involving multiple genes located in a cluster can be associated with disease.
An example of such a disease is Prader-Willi syndrome (PWS). Deletions
of chromosome 15q11-q13 affecting multiple genes have the recognizable
consequence of PWS, whereas mutations involving individual genes within
that cluster do not. Duplications of certain chromosomal regions increase
the "dosage" of the genes that are present, sometimes with phenotypic
consequences. One process in which this commonly occurs is malignancy.
Rearrangement
of chromosomes can have a number of pathogenic consequences. Translocations
occur when 2 chromosomes inappropriately come together and exchange genetic
material. The breakpoints of such translocations can occur in or near
genes and affect their expression. A number of disease-causing genes have
been identified by studying translocations in people with specific phenotypes,
including neurofibromatosis type I at 17q11.2. Translocations can also
place the upstream part of one gene into continuity with the downstream
part of another gene, resulting in the production of a "fusion protein."
The classic example of this process is termed the Philadelphia translocation
that fuses the BCR gene with the ABL oncogene, resulting
in chronic myeloid leukemia due to the unregulated expression of the activated
oncogene. (Fig. 1)
Translocations
may have effects on gene expression without physically disrupting a gene.
The genome is very heterogeneous in terms of its ability to support gene
expression. Chromosomal banding patterns represent this heterogeneity.
Gene-poor regions of the genome are generally found in areas that are
heterochromatic and are reflected in the G banding pattern of the chromosome.
Gene-rich regions are generally located in areas that are euchromatic
and are reflected in the R banding pattern. Placement of a normally euchromatic
gene into or adjacent to a heterochromatic region can alter the chromatin
environment of the gene, causing suppression of its expression. This phenomenon
is well characterized in model organisms such as Drosophila melanogaster
and is referred to as position effect variegation. A translocation that
places heterochromatin in the vicinity of a gene located in a euchromatic
region is now recognized as a mechanism that accounts for a number of
human genetic diseases. This type of indirect influence on gene regulation
is another example of epigenetic dysregulation of gene expression.
Other
epigenetic processes can cause human disease. Genomic imprinting is a
phenomenon characterized by silencing of a locus on a chromosome from
a specific parental origin. For example, the tumor suppressor CDKN1C
is active on the maternal chromosome 11p15.5 but silenced on the paternal
chromosome. Mutation of the paternally inherited gene has no obvious effect,
whereas mutation of the maternal copy causes the expression of the gene
to drop, not to the 50% level associated with nonimprinted loci, but to
drastically low levels, leading to a partial Beckwith-Wiedeman syndrome
phenotype. Other reported examples of epigenetic dysregulation, such as
alteration of regulatory methylation patterns in triplet repeat disorders,
are increasing the interest in epigenetic processes in human disease.
The mechanism by which triplet repeat mutations cause illnesses will be
reviewed in the next column.
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