In many of the techniques outlined above, no specific
information is gained about the exact nature of the alteration in
the DNA. In some cases, the change detected may turn out to
be a polymorphism that has no direct bearing on the condition
under investigation. The exception to this is the protein
truncation test (PTT), which detects mutations that shorten the
protein product and are therefore more likely to be pathogenic.
In chemical cleavage of mismatch analysis, particular types of
base mismatch are cleaved specifically by the different chemicals
employed; this yields limited information about the type of
change observed.
Saturday, April 11, 2009
making a copy of the DNA
The technique relies on making a copy of the DNA in
the presence of modified versions of the four bases (A, C, G,
and T) which are fluorescently labelled with their own specific
tag. The sequencing products are then separated with the use
of long polyacrylamide gels with a laser being used to
automatically detect the fluorescent molecules as they migrate.
A computer program is then used to generate the DNA
sequence. Recent improvements in DNA sequencing have seen
polyacrylamide gels being replaced by capillary columns
allowing the method to be further automated.
the presence of modified versions of the four bases (A, C, G,
and T) which are fluorescently labelled with their own specific
tag. The sequencing products are then separated with the use
of long polyacrylamide gels with a laser being used to
automatically detect the fluorescent molecules as they migrate.
A computer program is then used to generate the DNA
sequence. Recent improvements in DNA sequencing have seen
polyacrylamide gels being replaced by capillary columns
allowing the method to be further automated.
Hybridisation methods and “gene-chip” technology
In most of the methods described above, the specific site of a
mutation within a gene is not known until after DNA
sequencing has been completed. If the mutation is very
common, however, methods may be used that specifically
interrogate the site of the mutation. One of the simplest ways
of doing this is by using a restriction enzyme (see above);
however, this is not applicable in all situations.
mutation within a gene is not known until after DNA
sequencing has been completed. If the mutation is very
common, however, methods may be used that specifically
interrogate the site of the mutation. One of the simplest ways
of doing this is by using a restriction enzyme (see above);
however, this is not applicable in all situations.
use of DNA probe technology
Another possibility is the use of DNA probe technology.
This utilises the tendency of two complementary singlestranded
DNA molecules to anneal together to produce a
double-stranded duplex. This method involves the DNA under
investigation being immobilised onto a solid support such as
nylon. A labelled single-stranded DNA probe may then be used
to determine whether a specific sequence is present. This
technique is often referred to as forward dot-blotting.
This utilises the tendency of two complementary singlestranded
DNA molecules to anneal together to produce a
double-stranded duplex. This method involves the DNA under
investigation being immobilised onto a solid support such as
nylon. A labelled single-stranded DNA probe may then be used
to determine whether a specific sequence is present. This
technique is often referred to as forward dot-blotting.
labelled target DNA
Alternatively, the probes may be immobilised to the
membrane and hybridised with the labelled target DNA, that is
free in solution (the reverse dot-blot approach). It is this basic
principle that has been developed into the so-called “gene
chip” technology. In this technique, literally thousands of short
DNA probe molecules are first attached to silica-based support
materials. The DNA under investigation is then fluorescently
labelled and hybridised to the probe matrix. The large number
of probes used enables the pattern of hybridisation to be
translated into sequence information. At present, however, the
high cost of this approach means that it is of limited value for
the analysis of rare disease genes in a diagnostic setting.
membrane and hybridised with the labelled target DNA, that is
free in solution (the reverse dot-blot approach). It is this basic
principle that has been developed into the so-called “gene
chip” technology. In this technique, literally thousands of short
DNA probe molecules are first attached to silica-based support
materials. The DNA under investigation is then fluorescently
labelled and hybridised to the probe matrix. The large number
of probes used enables the pattern of hybridisation to be
translated into sequence information. At present, however, the
high cost of this approach means that it is of limited value for
the analysis of rare disease genes in a diagnostic setting.
Non-PCR based analysis
Not every gene can be studied using PCR. In some conditions,
the mutation itself is large, and may have even deleted the
entire gene. In other cases, the gene may be very rich in G and
C bases, which makes conventional PCR difficult. In these
situations, the older methods of analysis are invaluable,
although generally more time-consuming than PCR-based
methods.
the mutation itself is large, and may have even deleted the
entire gene. In other cases, the gene may be very rich in G and
C bases, which makes conventional PCR difficult. In these
situations, the older methods of analysis are invaluable,
although generally more time-consuming than PCR-based
methods.
Southern blotting
Although largely replaced by PCR-based methods, Southern
blotting is still necessary to detect relatively large changes in
the DNA that exceed the limits of PCR. Genomic DNA is first
cut using restriction enzymes and the digested fragments
fractionated using gel electrophoresis. The DNA is then
transferred by capillary blotting onto nylon membrane before
radiolabelled probes are used to investigate the region of
interest.
blotting is still necessary to detect relatively large changes in
the DNA that exceed the limits of PCR. Genomic DNA is first
cut using restriction enzymes and the digested fragments
fractionated using gel electrophoresis. The DNA is then
transferred by capillary blotting onto nylon membrane before
radiolabelled probes are used to investigate the region of
interest.
Pulse-field gel electrophoresis (PFGE)
In a development of standard Southern blotting methods,
PFGE uses specialised restriction enzymes and electrophoresis
conditions to fractionate the genomic DNA to a
high-resolution. This method is more applicable to the
detection of large deletions, well out of the range of PCR.
PFGE uses specialised restriction enzymes and electrophoresis
conditions to fractionate the genomic DNA to a
high-resolution. This method is more applicable to the
detection of large deletions, well out of the range of PCR.
Future developments
DNA sequencing currently provides information on the order
of bases within a gene to a high degree of accuracy. However,
the large size of many genes involved (e.g. the breast cancer
susceptibility genes BRCA1 and BRCA2) and the number of
patients requiring analysis means that improvements in
throughput are highly desirable. Robotic workstations are
currently being introduced into many molecular genetic
laboratories to try to meet this demand by automating many of
the laborious sample handling steps involved.
of bases within a gene to a high degree of accuracy. However,
the large size of many genes involved (e.g. the breast cancer
susceptibility genes BRCA1 and BRCA2) and the number of
patients requiring analysis means that improvements in
throughput are highly desirable. Robotic workstations are
currently being introduced into many molecular genetic
laboratories to try to meet this demand by automating many of
the laborious sample handling steps involved.
Human Genome Project
In addition to improvements in sample throughput,
molecular genetic laboratories are increasingly paying attention
to the functional significance of the genetic changes that they
detect. Functional studies are especially important in predictive
and pre-symptomatic analysis, where the relevance of a
mutation has a direct bearing on the decision making process.
The vast quantity of information that has been generated by
the Human Genome Project will undoubtedly increase the
ability to predict the effect of specific mutations. However,
there may well come a time when the detection of a genetic
event is only the first stage in the investigation into its
functional effect.
molecular genetic laboratories are increasingly paying attention
to the functional significance of the genetic changes that they
detect. Functional studies are especially important in predictive
and pre-symptomatic analysis, where the relevance of a
mutation has a direct bearing on the decision making process.
The vast quantity of information that has been generated by
the Human Genome Project will undoubtedly increase the
ability to predict the effect of specific mutations. However,
there may well come a time when the detection of a genetic
event is only the first stage in the investigation into its
functional effect.
Molecular analysis of mendelian disorders
Molecular genetic analysis is now possible for an increasing
number of single gene disorders. In some cases direct mutation
detection is feasible and molecular testing will provide or
confirm the diagnosis in the index case in a family. This enables
tests to be offered to other relatives to provide presymptomatic
diagnosis, carrier testing and prenatal diagnosis as appropriate.
For recessive conditions that are due to a small number of gene
mutations, or those that have a commonly occurring mutation,
it may also be possible to offer molecular based carrier tests to
an unrelated spouse. Tests for very rare disorders in the UK are
usually carried out on a national basis by designated
laboratories. For the more common disorders, genetic analysis is
undertaken in most of the regionally based NHS molecular
genetic laboratories. In this chapter, examples of some of these
common inherited disorders have been chosen to illustrate the
range of tests performed.
number of single gene disorders. In some cases direct mutation
detection is feasible and molecular testing will provide or
confirm the diagnosis in the index case in a family. This enables
tests to be offered to other relatives to provide presymptomatic
diagnosis, carrier testing and prenatal diagnosis as appropriate.
For recessive conditions that are due to a small number of gene
mutations, or those that have a commonly occurring mutation,
it may also be possible to offer molecular based carrier tests to
an unrelated spouse. Tests for very rare disorders in the UK are
usually carried out on a national basis by designated
laboratories. For the more common disorders, genetic analysis is
undertaken in most of the regionally based NHS molecular
genetic laboratories. In this chapter, examples of some of these
common inherited disorders have been chosen to illustrate the
range of tests performed.
haemoglobinopathies
The haemoglobinopathies are a heterogeneous group of
inherited disorders characterised by the absent, reduced or
altered expression of one or more of the globin chains of
haemoglobin. The globin gene clusters on chromosome 16
include two -globin genes and on chromosome 11 a -globin
gene. The haemoglobinopathies represent the commonest
single-gene disorders in the world population and have had
profound effects on the provision of health care in some
developing countries.
inherited disorders characterised by the absent, reduced or
altered expression of one or more of the globin chains of
haemoglobin. The globin gene clusters on chromosome 16
include two -globin genes and on chromosome 11 a -globin
gene. The haemoglobinopathies represent the commonest
single-gene disorders in the world population and have had
profound effects on the provision of health care in some
developing countries.
Various mutations
Various mutations in the -globin gene cause structural
alterations in haemoglobin, the most important being the point
mutation that produces haemoglobin S and causes sickle cell
disease. Direct detection of this point mutation permits carrier
detection and first-trimester prenatal diagnosis.
alterations in haemoglobin, the most important being the point
mutation that produces haemoglobin S and causes sickle cell
disease. Direct detection of this point mutation permits carrier
detection and first-trimester prenatal diagnosis.
Thalassaemias
The thalassaemias are due to a reduced rate of synthesis of
- or -globin chains, leading to an imbalance in their
production. -thalassaemia is a defect of -globin chain
synthesis. Each normal adult chromosome expresses two copies
of the -globin gene and disease severity is proportional to the
number of -globin genes lost following a mutational event. In
the most severe type, Barts hydrops fetalis, all four copies are
lost, leading to a severe phenotype associated with stillbirth or
early neonatal death. The -globin gene cluster contains a
number of repeat regions that increase the likelihood of
unequal crossover during meiosis. As a result, relatively large
deletions are the commonest type of mutations that give rise to
-thalassamia. In particular, a 3.7 kilobase (kb) deletion is
common in patients from Africa, the Mediterranean, Middle
East and India. A 4.2 kb deletion is common in patients from
southeast Asia and the Pacific Islands. Both 3.7 kb and 4.2 kb
deletions can be detected by PCR analysis; however, since
amplification of the region is often technically challenging,
Southern blotting is still considered a reliable method of
analysis.
- or -globin chains, leading to an imbalance in their
production. -thalassaemia is a defect of -globin chain
synthesis. Each normal adult chromosome expresses two copies
of the -globin gene and disease severity is proportional to the
number of -globin genes lost following a mutational event. In
the most severe type, Barts hydrops fetalis, all four copies are
lost, leading to a severe phenotype associated with stillbirth or
early neonatal death. The -globin gene cluster contains a
number of repeat regions that increase the likelihood of
unequal crossover during meiosis. As a result, relatively large
deletions are the commonest type of mutations that give rise to
-thalassamia. In particular, a 3.7 kilobase (kb) deletion is
common in patients from Africa, the Mediterranean, Middle
East and India. A 4.2 kb deletion is common in patients from
southeast Asia and the Pacific Islands. Both 3.7 kb and 4.2 kb
deletions can be detected by PCR analysis; however, since
amplification of the region is often technically challenging,
Southern blotting is still considered a reliable method of
analysis.
thalassaemia
thalassaemia results from a variety of molecular defects
that either reduce or completely abolish -globin synthesis. Over
200 mutations have so far been reported with point mutations
and small deletions comprising the majority. Although a large
number of mutations have been reported, the prevalence of
specific mutations is dependent on the ethnic origin. Diagnostic
testing therefore requires knowledge of the mutation spectrum
in the population being screened. Eighty per cent of mutations
that either reduce or completely abolish -globin synthesis. Over
200 mutations have so far been reported with point mutations
and small deletions comprising the majority. Although a large
number of mutations have been reported, the prevalence of
specific mutations is dependent on the ethnic origin. Diagnostic
testing therefore requires knowledge of the mutation spectrum
in the population being screened. Eighty per cent of mutations
Cystic fibrosis (CF)
Along with fragile X syndrome, CF represents the commonest
request for analysis to most molecular genetic laboratories,
because of the high frequency of carriers in the population (1 in
22 in the UK). The incidence of CF varies between approximately
1 in 2000 live births in white caucasians to 1 in 90 000 in asians.
request for analysis to most molecular genetic laboratories,
because of the high frequency of carriers in the population (1 in
22 in the UK). The incidence of CF varies between approximately
1 in 2000 live births in white caucasians to 1 in 90 000 in asians.
Cystic fibrosis
Cystic fibrosis is caused by mutations in the cystic fibrosis
transmembrane regulator (CFTR) gene located on the long
arm of chromosome 7, which contains 27 exons. Approximately
700 mutations have been described, many of which are
“private” mutations restricted to a particular lineage.
Approximately half the mutations are “mis-sense” (i.e. the
protein product is full length but contains an amino acid
substitution). The commonest single mutation in CF is a
deletion known as F508 that accounts for at least 70% of cystic
fibrosis mutations in northern Europeans.
transmembrane regulator (CFTR) gene located on the long
arm of chromosome 7, which contains 27 exons. Approximately
700 mutations have been described, many of which are
“private” mutations restricted to a particular lineage.
Approximately half the mutations are “mis-sense” (i.e. the
protein product is full length but contains an amino acid
substitution). The commonest single mutation in CF is a
deletion known as F508 that accounts for at least 70% of cystic
fibrosis mutations in northern Europeans.
molecular genetic laboratories
Most molecular genetic laboratories will test for the
commonest CF mutations using either an ARMS (amplification
refractory mutation system) PCR analysis or other
mutation-specific tests such as OLA (oligonucleotide ligation
assay). It should be remembered that since the frequency of
mutations varies between populations, the panel of mutations
tested in one ethnic group may be of less value in another
ethnic group and consequently knowledge of the mutation
spectrum in the local population is important.
commonest CF mutations using either an ARMS (amplification
refractory mutation system) PCR analysis or other
mutation-specific tests such as OLA (oligonucleotide ligation
assay). It should be remembered that since the frequency of
mutations varies between populations, the panel of mutations
tested in one ethnic group may be of less value in another
ethnic group and consequently knowledge of the mutation
spectrum in the local population is important.
Fragile X syndrome (FRAX–A)
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 15–20%
of all X linked mental retardation. Affected males have
moderate to severe mental retardation, whereas affected
females have milder retardation and phenotypic features.
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 15–20%
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
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.
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
The fragile site associated with FRAX–A 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
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
FRAX–A 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.
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.
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