Gene mapping and molecular pathology

The corresponding number of mapped genes
had risen to 928 by the ninth meeting in 1987 as molecular
techniques replaced those of traditional somatic cell genetics.
The total number of mapped X linked loci also rose, from 155
in 1973 to 308 in 1987. The number of mapped genes has
continued to increase rapidly since then, reflecting the
development of new molecular biological techniques and the
institution of the Human Genome Project.

Human Genome Project

The Human Genome Project was initiated in 1995 as an
international collaborative project with the aim of determining
the DNA sequence of each of the human chromosomes and of
providing unrestricted public access to this information.
Sequencing data have been submitted by 16 collaborating
centres: eight from the United States, three from Germany, two
from Japan and one from France, China, and the UK
respectively. The UK contribution came from the Sanger
Centre at Hinxton in Cambridgeshire, jointly funded by the
Wellcome Trust and the Medical Research Council.

human genome project

The human genome project consortium used a hierarchical
shotgun approach in which overlapping bacterial clones were
sequenced using mapping data from publicly available maps.
Each bacterial clone was analysed to provide sequence data
with 99.99% accuracy. The first draft of the human sequence
covering 90% of the gene-rich regions of the human genome
was published in a historic article in Nature in February 2001

Gene localisation

Prior to 1980, only a few genes, for disorders whose
biochemical basis was known, had been identified. With the
advent of molecular techniques the first step in isolating many
genes for human diseases was to locate their chromosomal
position by gene mapping studies. In some disorders, such as
Huntington disease, this was achieved by undertaking linkage
studies using polymorphic DNA markers in affected families,
without any prior information about which chromosome
carried the gene. In other disorders, the likely position of the
gene was suggested by identification of a chromosomal
rearrangement in an affected individual in whom it was likely
that one of the chromosomal break points disrupted the gene.
The neurofibromatosis type 1 (NF1) gene, for example, was
isolated after the identification of such a translocation followed
by cloning and sequencing of DNA from the region of the
break point on chromosome 17.

Duchenne muscular dystrophy

In Duchenne muscular dystrophy, several affected females
had been reported who had one X chromosome disrupted by
an X:autosome translocation with the normal X chromosome
being preferentially inactivated. The site of the break point in
these cases was always on the short arm of the X chromosome
at Xp21, which suggested that this was the location of the gene
for DMD. DNA variations in this region, identified by
hybridisation with DNA probes, provided markers that were
shown to be linked to the gene for DMD in family studies in
1983. Strategies were then developed to identify DNA
sequences from the region of the gene for DMD, some of which
were missing in affected boys indicating that they represented
deleted intragenic sequences. The entire gene for DMD was
subsequently cloned in 1987 and its structure determined.

Gene tracking

Once a disease gene has been located using linkage analysis,
DNA markers can be used to track the disease gene through
families to predict the genetic state of individuals at risk. Prior
to identifying specific gene mutations, this can provide
information about carrier risk and enable prenatal diagnosis in
certain situations. Before gene tracking can be used to provide
a predictive test, family members known to be affected or
unaffected must be tested to find an informative DNA marker
within the family and to identify which allele is segregating with
the disease gene in that particular kindred. Because
recombination occurs between homologous chromosomes at
meiosis, a DNA marker that is not very close to a gene on a
particular chromosome will sometimes be inherited
independently of the gene. The closer the marker is to a gene,
the less likely it is that recombination will occur. In practice,
markers that have shown less than 5% recombination with a
disease gene have been useful in detecting carriers and in
prenatal diagnosis, although there is always a margin of error
with this type of test and results are quoted as a probability of
carrying the gene and not as a definitive result. Linkage studies
using intragenic markers provide much more accurate
prediction of genetic state, but this approach is only used now
when mutation analysis is not possible, as in some cases of
Duchenne muscular dystrophy, Marfan syndrome and
neurofibromatosis type 1.

Gene identification

Once the chromosomal location of a gene has been identified,
there are several strategies that can be employed to isolate the
gene itself. Genes within the region of interest can be searched
for by using techniques such as cDNA selection and screening,
CpG island identification and exon trapping. Any genes
identified can then be studied for mutations in affected
individuals. Alternatively, candidate genes can be identified by
their function or expression patterns or by sequence homology
with genes known to cause similar phenotypes in animals. The
gene for Waardenburg syndrome, for example, was localised to
chromosome 2q by linkage studies and the finding of a
chromosomal abnormality in an affected subject. Identification
of the gene was then aided by recognition of a similar
phenotype in splotch mice. Mutations in the PAX3 gene were
found to underlie the phenotype in both mice and humans.

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, 5–10% of
mutations are due to duplication of exons within the
dystrophin gene, and in Charcot–Marie–Tooth 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 of LINE1 repetitive
sequences into the F8C gene.

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.

Frameshift mutations

Mutations that remove or add a number of bases that are not a
multiple of three will result in an alteration of the transcription
and translation reading frames. These mutations result in the
translation of an abnormal protein from the site of the
mutation onwards and almost always result in the generation of
a premature stop codon. In Duchenne muscular dystrophy,
most deletions alter the reading frame, leading to lack of
production of a functional dystrophin protein and a severe
phenotype. In Becker muscular dystrophy, most deletions
maintain the correct reading frame, leading to the production
of an internally truncated dystrophin protein that retains some
function and results in a milder phenotype.

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 20–35 in the
normal population to 40–80 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
Prader–Willi syndromes.

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 in
familial 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.

different mutation types

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 neuronal
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 thePMP22 gene, with an increase in gene product, results in
Charcot–Marie–Tooth disease type 1a. Interestingly, point
mutations in the same gene produce a similar phenotype by
functioning as activating mutations.