Types of mutation

In a few genetic diseases, all affected individuals have the same mutation. In sickle cell
disease, for example, all mutant genes have a single base substitution, changing the sixth
codon of the beta-globin gene from GAG to GTG, resulting in the substitution of valine for
glutamic acid. In Huntington disease, all affected individuals have an expansion of a CAG
trinucleotide repeat expansion. The majority of mendelian disorders are, however, due to
many different mutations in a single gene. In some cases, one or more mutations are
particularly frequent. In cystic fibrosis, for example, over 700 mutations have been described,
but one particular mutation, _F508, accounts for about 70% of all cases in northern
Europeans. In many conditions, the range of mutations observed is very variable. In DMD,
for example, mutations include deletions, duplications and point mutations.
Deletions
Large gene deletions are the causal mutations in several disorders including thalassaemia,
haemophilia A and Duchenne muscular dystrophy. In some cases the entire gene is deleted,
as in -thalassaemia; in others, there is only a partial gene deletion, as in Duchenne muscular
dystrophy.
Duplications and insertions
Pathological duplication mutations are observed in some disorders. In Duchenne muscular
dystrophy, 510% of mutations are due to duplication of exons within the dystrophin gene,
and in CharcotMarieTooth disease type 1a, 70% of mutations involve duplication of the
entire PMP22 gene. In DMD the mutation acts by causing a shift in the translation reading
frame, and in CMT 1a by increasing the amount of gene product produced. Insertions of
foreign DNA sequences into a gene also disrupt its function, as in haemophilia A caused by
insertion
Trinucleotide repeat expansions
Expanded trinucleotide repeat regions represent new, unstable mutations that were
identified in 1991. This type of mutation is the cause of several major genetic disorders,
including fragile X syndrome, myotonic dystrophy, Huntington disease, spinocerebellar
ataxia and Friedreich ataxia. In the normal copies of these genes the number of repeats of the
trinucleotide sequence is variable. In affected individuals the number of repeats expands
outside the normal range. In Huntington disease the expansion is small, involving a doubling
of the number of repeats from 2035 in the normal population to 4080 in affected
individuals.
In fragile X syndrome and myotonic dystrophy the expansion may be very large, and
the size of the expansion is often very unstable when transmitted from affected parent to
child. Severity of these disorders correlates broadly with the size of the expansion: larger
expansions causing more severe disease.

Epigenetic effects
Epigenetic effects are inherited molecular changes that do not alter DNA sequence.
These can affect the expression of genes or the function of the protein product. Epigenetic
effects include DNA methylation and alteration of chromatin configuration or protein
conformation. Methylation of controlling elements silences gene expression as a normal
event during development. Abnormalities of methylation may result in genetic disease.
In fragile X syndrome, methylation of the promotor occurs when there is a large CGG
expansion, inactivating the gene and causing the clinical phenotype. Methylation is also
involved in the imprinting of certain genes, where abnormalities lead to disorders such as
Angelman and PraderWilli syndromes.

Point mutations
Most disease-causing mutations are simple base substitutions, which can have variable effect.
Mis-sense mutations result in the replacement of one amino acid with another in the protein
product and have an effect when an essential amino acid is involved. Non-sense mutations
result in replacement of an amino acid codon with a stop codon. This often results in mRNA
instability, so that no protein product is produced. Other single base substitutions may alter
the splicing of exons and introns, or affect sequences involved in regulating gene expression
such as gene promoters or polyadenylation sites.

Modifier genes
The variation in phenotype between different affected members of the same
family who have identical gene mutations may be due in part to environmental
factors, but is probably also determined by the presence or absence of particular
alleles at other loci, referred to as modifier genes. Modifying genes may for
example, determine the incidence of complications in insulin dependent
diabetes, the development of amyloidosis infamilial Mediterranean fever and
the occurrence of meconium ileus in cystic fibrosis.

Abnormalities of gene function

Different types of genetic mutation have different consequences for gene function. The
effects on phenotype may reflect either loss or gain of function. In some genes, either type of
mutation may occur, resulting in different phenotypes.

Loss of function mutations

Loss of function mutations result in reduced or absent function of the gene product.
This type of mutation is the most common, and generally results in a recessive phenotype, in
which heterozygotes with 50% of normal gene activity are unaffected, and only homozygotes
with complete loss of function are clinically affected. Occasionally, loss of function
mutations may have a dominant effect. Heterozygosity for chromosomal deletions usually
causes an abnormal phenotype and this is probably due to haploinsufficiency of a number of
genes.
Many different mutation types can result in loss of function of the gene product and
when a variety of mutations in a gene cause a single phenotype, these are all likely to
represent loss of function mutations. In fragile X syndrome, for example, the most common
mutation is a pathological expansion of a CGG trinucleotide repeat that silences the FMR1
gene. Occasionally the syndrome is due to a point mutation in the FMR1 gene, also
associated with lack of the gene product that produces the same phenotype.

Dominant negative effect

In some conditions, the abnormal gene product not only loses normal function but also
interferes with the function of the product from the normal allele. This type of mutation acts
in a dominant fashion and is referred to as having a dominant negative effect. In type I
osteogenesis imperfecta (OI), for example, the causal mutations in the COL1A1 and COL1A2
genes produce an abnormal type I collagen that interferes with normal triple helix formation,
resulting in production of an abnormal mature collagen responsible for the OI phenotype.

Gain of function mutation

When the protein product produced by a mutant gene acquires a completely novel function,
the mutation is referred to as having a gain of function effect. These mutations usually result
in dominant phenotypes because of the independent action of the gene product. The CAG
repeat expansions in Huntington disease and the spinocerebellar ataxias exert a gain of
function effect, by resulting in the incorporation of elongated polyglutamine tracts in the
protein products. This causes formation of intracellular aggregates that result in neurona cell
death. Mutations producing a gain of function effect are likely to be very specific and other
mutations in the same gene are unlikely to produce the same phenotype. In the androgen
receptor gene, for example, a trinucleotide repeat expansion mutation results in the phenotype
of spinobulbar muscular atrophy (Kennedy syndrome), whereas a point mutation leading to
loss of function results in the completely different phenotype of testicular feminisation
syndrome.

Overexpression
Overexpression of a structurally normal gene may occasionally produce an abnormal
phenotype. Complete duplication of the PMP22 gene, with an increase in gene product,
results in CharcotMarieTooth disease type 1a. Interestingly, point mutations in the same
gene produce a similar phenotype by functioning as activating mutations. Although examples
of gene duplication are not common, the abnormal phenotype associated with chromosomal
duplications is probably due to the overexpression of a number of genes.

Fragile X syndrome (FRAXA)

Fragile X syndrome is one of a group of disorders caused by the expansion of a triplet
repeat region within a gene. It is associated with the presence of a fragile site on the X
chromosome (Xq27.3), categorised as FRAX-A. The syndrome is characterised by mental
retardation and accounts for 1520% of all X linked mental retardation. Affected males have
moderate to severe mental retardation, whereas affected females have milder retardation and
phenotypic features. Fragile X syndrome is caused by an expanded CGG repeat in the
untranslated region of the FMR-1 gene, which results in reduction or abolition of expression
of the gene by methylation of the gene promoter. In normal individuals, the number of CGG
repeats varies between 6 and 54 units and is stably inherited. However, if individuals have
between 55 and 200 repeats (although apparently unaffected), there is an increased risk of the
repeat region expanding further into the full mutation range (200 repeats) that is associated
with mental retardation.
The fragile site associated with FRAXA may be detected using cytogenetic methods
by culturing cells in the absence of folic acid and thymidine but this is not a sensitive test for
detecting carrier females. The expansion of the CGG repeat in the FMR-1 gene may be
detected at the DNA level using PCR. After amplification, the size of the repeat from each
chromosomal copy is determined by polyacrylamide gel electrophoresis. Samples with a
known number of repeats are used as size standards. This type of approach can be used only
as a screen to detect normal sized alleles. Because full mutations with long stretches of CGG
repeats are too large to amplify effectively, Southern blotting is still widely used in FRAXA
analysis. This method can also be modified to determine the methylation status of the gene
(the main influence on normal FMR-1 gene expression). In prenatal diagnosis, methylation
analysis is problematic owing to the presence of fetal methylation patterns, and the size of the
repeat becomes the most reliable predictive indicator.