HD is a progressive fatal neurodegenerative disease. Like
FRAX–A, HD is caused by a triplet repeat expansion. The HD
expansion involves a CAG triplet in exon 1 of the IT15 gene on
chromosome 4. The expansion is translated into a
polyglutamine tract in the huntingtin protein gene product
that is believed to cause a dominant gain of function leading to
neuronal loss.
In normal individuals, the CAG unit in exon 1 has between
9 and 35 repeats. Affected individuals have repeats of 36 units
or greater, with over 90% of affected subjects having 40–55
repeats. In general, the greater the number of repeats an
individual has, the earlier the age of onset will be, although this
relationship is stronger for higher repeat numbers.
Saturday, April 11, 2009
CAG
Since the CAG repeat expansion is the sole mutation
responsible for all HD cases, molecular genetic analysis
concentrates on this single region. Small CAG expansions can
be detected using PCR amplification of the repeat region.
The PCR products are then sized using polyacrylamide gel
electrophoresis. Samples with known repeat sizes may be used
as controls to determine the size of the expansion. Larger
expansions cannot be detected by PCR and the time-consuming
Southern blotting method must be used in cases where two
normal sized repeat alleles are not detected by PCR.
responsible for all HD cases, molecular genetic analysis
concentrates on this single region. Small CAG expansions can
be detected using PCR amplification of the repeat region.
The PCR products are then sized using polyacrylamide gel
electrophoresis. Samples with known repeat sizes may be used
as controls to determine the size of the expansion. Larger
expansions cannot be detected by PCR and the time-consuming
Southern blotting method must be used in cases where two
normal sized repeat alleles are not detected by PCR.
Charcot–Marie–Tooth disease (CMT)
CMT disease (or hereditary motor and sensory neuropathy,
HMSN) is clinically and genetically heterogeneous, but is
generally characterised by wasting and weakness of the distal
limb muscles with or without distal sensory loss. CMT may be
inherited in an autosomal dominant, autosomal recessive or
X linked manner. Clinically, the condition is divided into the
demyelinating CMT1 (with reduced nerve conduction
velocities) and axonal CMT2 (with nerve conduction velocites
largely preserved). Rarer clinical forms exist, including the
severe Dejerine–Sottas syndrome and hereditary neuropathy
with increased reflexes. The related condition HNPP
(hereditary neuropathy with liability to pressure palsies) creates
a milder phenotype characterised by recurrent, usually
transient sensorimotor neuropathies.
HMSN) is clinically and genetically heterogeneous, but is
generally characterised by wasting and weakness of the distal
limb muscles with or without distal sensory loss. CMT may be
inherited in an autosomal dominant, autosomal recessive or
X linked manner. Clinically, the condition is divided into the
demyelinating CMT1 (with reduced nerve conduction
velocities) and axonal CMT2 (with nerve conduction velocites
largely preserved). Rarer clinical forms exist, including the
severe Dejerine–Sottas syndrome and hereditary neuropathy
with increased reflexes. The related condition HNPP
(hereditary neuropathy with liability to pressure palsies) creates
a milder phenotype characterised by recurrent, usually
transient sensorimotor neuropathies.
genes involved in CMT
Many of the genes involved in CMT have now been cloned
and sequenced, allowing a genetic classification to be made
depending on the mutation or gene locus identified. Mutations
in over five genes have been reported in CMT, including
PMP22 (peripheral myelin protein on chromosome 17),
MPZ (myelin protein zero on chromosome 1), Connexin-32
(X chromosome), EGR2 and NEFL. The commonest mutational
event is the duplication of the entire PMP22 gene resulting in
clinical CMT type 1a. A deletion of the same gene gives rise to
the milder HNPP phenotype. Phenotypes of varying severity
can also be produced by point mutations (often base
substitutions) in any of the five genes mentioned above.
Prediction of disease severity in presymptomatic patients is
difficult as there is varying severity even within families.
and sequenced, allowing a genetic classification to be made
depending on the mutation or gene locus identified. Mutations
in over five genes have been reported in CMT, including
PMP22 (peripheral myelin protein on chromosome 17),
MPZ (myelin protein zero on chromosome 1), Connexin-32
(X chromosome), EGR2 and NEFL. The commonest mutational
event is the duplication of the entire PMP22 gene resulting in
clinical CMT type 1a. A deletion of the same gene gives rise to
the milder HNPP phenotype. Phenotypes of varying severity
can also be produced by point mutations (often base
substitutions) in any of the five genes mentioned above.
Prediction of disease severity in presymptomatic patients is
difficult as there is varying severity even within families.
duplication or deletion of the PMP22 gene
Detection of the duplication or deletion of the PMP22 gene
is achieved using fluorescent dosage PCR analysis to determine
the number of gene copies present. Following initial PCR
amplification with fluorescently-labelled primers, the products
are analysed by automated laser-induced fluorescence. Point
mutations in all five CMT genes are detected by a variety of
methods depending on local practices, including SSCP, DGGE
and DNA sequencing. Requests for prenatal testing in the UK
are rare.
is achieved using fluorescent dosage PCR analysis to determine
the number of gene copies present. Following initial PCR
amplification with fluorescently-labelled primers, the products
are analysed by automated laser-induced fluorescence. Point
mutations in all five CMT genes are detected by a variety of
methods depending on local practices, including SSCP, DGGE
and DNA sequencing. Requests for prenatal testing in the UK
are rare.
Spinal muscular atrophy (SMA)
SMA encompasses a clinically and genetically heterogeneous
group of disorders characterised by degeneration and loss of
the anterior horn cells in the spinal cord and sometimes in the
brainstem nuclei, resulting in muscle weakness and atrophy.
Most cases are inherited in an autosomal recessive fashion,
although some affected families show dominant inheritance.
Childhood onset SMA is the second most common, lethal
autosomal recessive disorder in white populations, with an
overall incidence of 1 in 10 000 live births and a carrier
frequency of approximately 1 in 50. It is estimated to be the
second most frequent disease seen in paediatric neuromuscular
clinics after Duchenne muscular dystrophy.
group of disorders characterised by degeneration and loss of
the anterior horn cells in the spinal cord and sometimes in the
brainstem nuclei, resulting in muscle weakness and atrophy.
Most cases are inherited in an autosomal recessive fashion,
although some affected families show dominant inheritance.
Childhood onset SMA is the second most common, lethal
autosomal recessive disorder in white populations, with an
overall incidence of 1 in 10 000 live births and a carrier
frequency of approximately 1 in 50. It is estimated to be the
second most frequent disease seen in paediatric neuromuscular
clinics after Duchenne muscular dystrophy.
Childhood onset SMA
Childhood onset SMA can be classified into three types,
distinguished on the basis of clinical severity and age of onset.
In type I (Werdnig–Hoffman disease), onset occurs within the
first six months of life and children usually die within two years.
In type II (intermediate type Dubowitz disease) onset is before
18 months with death occuring after two years. In type III
(Kugelberg–Welander disease), the disease has a later onset
and milder, chronic cause with affected children achieving
ambulation.
distinguished on the basis of clinical severity and age of onset.
In type I (Werdnig–Hoffman disease), onset occurs within the
first six months of life and children usually die within two years.
In type II (intermediate type Dubowitz disease) onset is before
18 months with death occuring after two years. In type III
(Kugelberg–Welander disease), the disease has a later onset
and milder, chronic cause with affected children achieving
ambulation.
SMN gene
At least three genes have been reported to be associated
with the SMA type I phenotype on chromosome 5, namely
SMN, NAIP and p44. Diagnostic analysis in SMA patients is
restricted largely to analysis of the SMN gene. The SMN gene is
present in two copies, one centromeric (SMNC) and one
telomeric (SMNT). The absence of exons 7 and 8 in both
copies of the SMNT gene is a very reliable diagnostic test for
the majority of patients, confirming the clinical diagnosis of
SMA. Point mutations have been detected in affected
individuals who do not have homozygous deletions. The
PCR-based assay used for determining the presence or absence
of the SMNT gene is not able to detect individuals who are
heterozygous deletion carriers, and a gene dosage method of
analysis has been developed to improve carrier detection.
with the SMA type I phenotype on chromosome 5, namely
SMN, NAIP and p44. Diagnostic analysis in SMA patients is
restricted largely to analysis of the SMN gene. The SMN gene is
present in two copies, one centromeric (SMNC) and one
telomeric (SMNT). The absence of exons 7 and 8 in both
copies of the SMNT gene is a very reliable diagnostic test for
the majority of patients, confirming the clinical diagnosis of
SMA. Point mutations have been detected in affected
individuals who do not have homozygous deletions. The
PCR-based assay used for determining the presence or absence
of the SMNT gene is not able to detect individuals who are
heterozygous deletion carriers, and a gene dosage method of
analysis has been developed to improve carrier detection.
Duchenne and Becker muscular dystrophies
Duchenne muscular dystrophy (DMD) and the milder Becker
form (BMD) are X linked recessive disorders causing
progressive proximal muscle weakness, associated with elevation
of serum creatine kinase levels. Weakness of the diaphragm and
intercostal muscles leads to respiratory insufficiency, and
involvement of the myocardium causing dilated
cardiomyopathy is common.
Both DMD and BMD result from mutations in the gene
encoding dystrophin, located at Xp21. The gene is one of the
largest identified covering approximately 2.5 megabases of
DNA and having 79 exons. Two-thirds of cases are caused by
deletion of one or more of the dystrophin exons that cluster in
two hot-spots within the gene. Large duplications account for a
further 5–10% of cases. The remainder of cases are due to a
variety of point mutations.
form (BMD) are X linked recessive disorders causing
progressive proximal muscle weakness, associated with elevation
of serum creatine kinase levels. Weakness of the diaphragm and
intercostal muscles leads to respiratory insufficiency, and
involvement of the myocardium causing dilated
cardiomyopathy is common.
Both DMD and BMD result from mutations in the gene
encoding dystrophin, located at Xp21. The gene is one of the
largest identified covering approximately 2.5 megabases of
DNA and having 79 exons. Two-thirds of cases are caused by
deletion of one or more of the dystrophin exons that cluster in
two hot-spots within the gene. Large duplications account for a
further 5–10% of cases. The remainder of cases are due to a
variety of point mutations.
types of mutations
Since just about all types of mutations can be seen in
DMD/BMD cases, a variety of techniques need to be used to
carry out a comprehensive molecular analysis. A multiplex PCR
approach in which a number of segments of the gene are
amplified simultaneously has been developed to rapidly detect
deletion of exons in males. Fluorescent dosage analysis can be
used to detect deletions and duplications in both affected males
and female carriers and chromosomal analysis using fluorescence
in situ hybridisation (FISH) techniques will also detect deletions
in female carriers. Detecting point mutations is possible with a
variety of methods including SSCP analysis, DGGE analysis, and
DNA sequencing but is not routine because of the very large
number of exons in the gene. In cases where the underlying
mutation is unknown, carrier detection and prenatal diagnosis
may still be accomplished by linkage analysis with a combination
of intragenic DNA markers and markers flanking the gene.
DMD/BMD cases, a variety of techniques need to be used to
carry out a comprehensive molecular analysis. A multiplex PCR
approach in which a number of segments of the gene are
amplified simultaneously has been developed to rapidly detect
deletion of exons in males. Fluorescent dosage analysis can be
used to detect deletions and duplications in both affected males
and female carriers and chromosomal analysis using fluorescence
in situ hybridisation (FISH) techniques will also detect deletions
in female carriers. Detecting point mutations is possible with a
variety of methods including SSCP analysis, DGGE analysis, and
DNA sequencing but is not routine because of the very large
number of exons in the gene. In cases where the underlying
mutation is unknown, carrier detection and prenatal diagnosis
may still be accomplished by linkage analysis with a combination
of intragenic DNA markers and markers flanking the gene.
Familial breast cancer
Breast cancer is the commonest cancer seen in young women
from developed countries, affecting about 20% of all women
who die of cancer. Although the majority of breast cancer cases
are sporadic, approximately 5% have an inherited component.
The two susceptibility genes identified so far are BRCA1 and
BRCA2. The BRCA1 gene on chromosome 17q21 is involved in
45–50% of inherited breast-only cancer and 75–80% of
inherited breast/ovarian cancer. The BRCA2 gene on
chromosome 13q12-13 is involved in approximately 35% of
inherited breast-only cancer and 20% of breast/ovarian cancer.
In addition, BRCA2 is involved in a significant proportion of
male breast cancer.
from developed countries, affecting about 20% of all women
who die of cancer. Although the majority of breast cancer cases
are sporadic, approximately 5% have an inherited component.
The two susceptibility genes identified so far are BRCA1 and
BRCA2. The BRCA1 gene on chromosome 17q21 is involved in
45–50% of inherited breast-only cancer and 75–80% of
inherited breast/ovarian cancer. The BRCA2 gene on
chromosome 13q12-13 is involved in approximately 35% of
inherited breast-only cancer and 20% of breast/ovarian cancer.
In addition, BRCA2 is involved in a significant proportion of
male breast cancer.
BRCA1 and BRCA2 gene
Both BRCA1 and BRCA2 genes are large, containing 24 and
26 exons respectively. Since being isolated, a considerable
number of mutations have been described in both genes – over
250 in BRCA1 and over 100 in BRCA2. Up to 90% of these
mutations are predicted to produce a truncated protein. This
makes it possible to screen for mutations in the large central
exon 11 using the protein truncation test. The remaining exons
are generally screened one-by-one using methods such as
SSCP/heteroduplex analysis or DNA sequencing.
Population-specific founder mutations have been found in
eastern European, Ashkenazi Jewish and Icelandic
populations. Screening for the common mutation is therefore
undertaken as the first step in investigating families from
these population groups.
26 exons respectively. Since being isolated, a considerable
number of mutations have been described in both genes – over
250 in BRCA1 and over 100 in BRCA2. Up to 90% of these
mutations are predicted to produce a truncated protein. This
makes it possible to screen for mutations in the large central
exon 11 using the protein truncation test. The remaining exons
are generally screened one-by-one using methods such as
SSCP/heteroduplex analysis or DNA sequencing.
Population-specific founder mutations have been found in
eastern European, Ashkenazi Jewish and Icelandic
populations. Screening for the common mutation is therefore
undertaken as the first step in investigating families from
these population groups.
Treatment of Genetic Disorders
The prevention of inherited disease by means of genetic and
reproductive counselling and prenatal diagnosis is often
emphasised. Genetic disorders may, however, be amenable to
treatment, either symptomatic or potentially curative.
Treatment may range from conventional drug or dietary
management and surgery to the future possibility of gene
therapy. The level at which therapeutic intervention can be
applied is influenced by the state of knowledge about the
primary genetic defect, its effect, its interaction with
environmental factors, and the way in which these may be
modified.
reproductive counselling and prenatal diagnosis is often
emphasised. Genetic disorders may, however, be amenable to
treatment, either symptomatic or potentially curative.
Treatment may range from conventional drug or dietary
management and surgery to the future possibility of gene
therapy. The level at which therapeutic intervention can be
applied is influenced by the state of knowledge about the
primary genetic defect, its effect, its interaction with
environmental factors, and the way in which these may be
modified.
Conventional treatment
Increasing knowledge of the molecular and biochemical basis
of genetic disorders will lead to better prospects for therapeutic
intervention and even the possibility of prenatal treatment in
some disorders. In the future, treatment of common
multifactorial disorders may be improved if genotype analysis of
affected individuals identifies those who are likely to respond to
particular drugs. In most single gene disorders, the primary
defect is not yet amenable to specific treatment. Conventional
treatment aimed at relieving the symptoms and preventing
complications remains important and may require a
multidisciplinary approach. Management of Duchenne
muscular dystrophy, for example, includes neurological and
orthopaedic assessment and treatment, physiotherapy,
treatment of chest infections and heart failure, mobility aids,
home modifications, appropriate schooling, and support for
the family, all of which aim to lessen the burden of the
disorder. Lay organisations often provide additional support for
the patients and their families. The Muscular Dystrophy
Organisation, for example, provides information leaflets,
supports research, and employs family care officers who work
closely with families and the medical services.
of genetic disorders will lead to better prospects for therapeutic
intervention and even the possibility of prenatal treatment in
some disorders. In the future, treatment of common
multifactorial disorders may be improved if genotype analysis of
affected individuals identifies those who are likely to respond to
particular drugs. In most single gene disorders, the primary
defect is not yet amenable to specific treatment. Conventional
treatment aimed at relieving the symptoms and preventing
complications remains important and may require a
multidisciplinary approach. Management of Duchenne
muscular dystrophy, for example, includes neurological and
orthopaedic assessment and treatment, physiotherapy,
treatment of chest infections and heart failure, mobility aids,
home modifications, appropriate schooling, and support for
the family, all of which aim to lessen the burden of the
disorder. Lay organisations often provide additional support for
the patients and their families. The Muscular Dystrophy
Organisation, for example, provides information leaflets,
supports research, and employs family care officers who work
closely with families and the medical services.
Environmental modification
The effects of some genetic disorders may be minimised by
avoiding or reducing exposure to adverse environmental
factors. These environmental effects are well recognised in
common disorders such as coronary heart disease, and
individuals known to be at increased genetic risk should be
encouraged to make appropriate lifestyle changes. Single gene
disorders may also be influenced by exposure to environmental
triggers. Attacks of acute intermittent porphyria can be
precipitated by drugs such as anticonvulsants, oestrogens,
barbiturates and sulphonamides, and these should be avoided
in affected individuals. Attacks of porphyria cutanea tarda are
precipitated by oestrogens and alcohol. In individuals with
glucose-6-phosphate dehydrogenase deficiency, drugs such as
primaquine and dapsone, as well as ingesting fava beans, cause
haemolysis.
avoiding or reducing exposure to adverse environmental
factors. These environmental effects are well recognised in
common disorders such as coronary heart disease, and
individuals known to be at increased genetic risk should be
encouraged to make appropriate lifestyle changes. Single gene
disorders may also be influenced by exposure to environmental
triggers. Attacks of acute intermittent porphyria can be
precipitated by drugs such as anticonvulsants, oestrogens,
barbiturates and sulphonamides, and these should be avoided
in affected individuals. Attacks of porphyria cutanea tarda are
precipitated by oestrogens and alcohol. In individuals with
glucose-6-phosphate dehydrogenase deficiency, drugs such as
primaquine and dapsone, as well as ingesting fava beans, cause
haemolysis.
genetic disorders.
Exposure to anaesthetic agents may be hazardous in some
genetic disorders. Myotonic dystrophy is associated with
increased anaesthetic risk and suxamethonium must not be
given to people with pseudocholinesterase deficiency.
Malignant hyperthermia (MH) is an autosomal dominant
condition in which individuals with MH susceptibility, who are
otherwise healthy, may develop life-threatening hyperpyrexial
reactions when exposed to a variety of inhalational anaesthetics
and muscle relaxants. Relatives with MH susceptibility can be
identified by muscle biopsy and in vitro muscle contracture
testing. This enables them to ensure that they are not exposed
to the triggering agents in any future anaesthetic. It is
recommended that susceptible individuals wear a MedicAlert or
similar medical talisman containing written information at all
times.
Exposure to sunlight precipitates skin fragility and blistering
in all the porphyrias except the acute intermittent form.
Sunlight should also be avoided in xeroderma pigmentosum (a
rare defect of DNA repair) and in oculocutaneous albinism
because of the increased risk of skin cancer.
genetic disorders. Myotonic dystrophy is associated with
increased anaesthetic risk and suxamethonium must not be
given to people with pseudocholinesterase deficiency.
Malignant hyperthermia (MH) is an autosomal dominant
condition in which individuals with MH susceptibility, who are
otherwise healthy, may develop life-threatening hyperpyrexial
reactions when exposed to a variety of inhalational anaesthetics
and muscle relaxants. Relatives with MH susceptibility can be
identified by muscle biopsy and in vitro muscle contracture
testing. This enables them to ensure that they are not exposed
to the triggering agents in any future anaesthetic. It is
recommended that susceptible individuals wear a MedicAlert or
similar medical talisman containing written information at all
times.
Exposure to sunlight precipitates skin fragility and blistering
in all the porphyrias except the acute intermittent form.
Sunlight should also be avoided in xeroderma pigmentosum (a
rare defect of DNA repair) and in oculocutaneous albinism
because of the increased risk of skin cancer.
Surgical management
Surgery plays an important role in various genetic disorders.
Many primary congenital malformations are amenable to
successful surgical correction. The presence of structural
abnormalities is often identified by prenatal ultrasound
scanning, and this allows arrangements to be made for delivery
to take place in a unit with the necessary neonatal surgical
facilities when this is likely to be required. In a few instances,
birth defects such as posterior urethral valves, may be amenable
to prenatal surgical intervention. In some disorders surgery
may be required for abnormalities that are secondary to an
underlying metabolic disorder. In girls with congenital adrenal
hyperplasia, virilisation of the external genitalia is secondary to
excess production of androgenic steroids in utero and requires
reconstructive surgery. In other disorders, structural
complications may occur later, such as the aortic dilatation that
may develop in Marfan syndrome. Surgery may also be needed
in genetic disorders that predispose to neoplasia, such as the
multiple endocrine neoplasia syndromes, where screening
family members at risk permits early intervention and improves
prognosis. Some women who carry mutations in the BRCA1 or
BRCA2 breast cancer genes elect to undergo prophylactic
mastectomy because of their high risk of developing breast
cancer.
Many primary congenital malformations are amenable to
successful surgical correction. The presence of structural
abnormalities is often identified by prenatal ultrasound
scanning, and this allows arrangements to be made for delivery
to take place in a unit with the necessary neonatal surgical
facilities when this is likely to be required. In a few instances,
birth defects such as posterior urethral valves, may be amenable
to prenatal surgical intervention. In some disorders surgery
may be required for abnormalities that are secondary to an
underlying metabolic disorder. In girls with congenital adrenal
hyperplasia, virilisation of the external genitalia is secondary to
excess production of androgenic steroids in utero and requires
reconstructive surgery. In other disorders, structural
complications may occur later, such as the aortic dilatation that
may develop in Marfan syndrome. Surgery may also be needed
in genetic disorders that predispose to neoplasia, such as the
multiple endocrine neoplasia syndromes, where screening
family members at risk permits early intervention and improves
prognosis. Some women who carry mutations in the BRCA1 or
BRCA2 breast cancer genes elect to undergo prophylactic
mastectomy because of their high risk of developing breast
cancer.
Metabolic manipulation
Some inborn errors of metabolism due to enzyme deficiencies
can be treated effectively. Although direct replacement of the
missing enzyme is not generally possible, enzyme activity can
be enhanced in some disorders. For example, phenobarbitone
induces hepatic glucuronyl transferase activity and may lower
circulating concentrations of unconjugated bilirubin in the
Crigler–Najjar syndrome type 2. Vitamins act as cofactors in
certain enzymatic reactions and can be effective if given in doses
above the usual physiological requirements. For example,
homocystinuria may respond to treatment with vitamin B6,
certain types of methylmalonic aciduria to vitamin B12, and
multiple carboxylase deficiency to biotin. It may also be possible
to stimulate alternate metabolic pathways. For example,
thiamine may permit a switch to pyruvate metabolism by means
of pyruvate dehydrogenase in pyruvate carboxylase deficiency.
can be treated effectively. Although direct replacement of the
missing enzyme is not generally possible, enzyme activity can
be enhanced in some disorders. For example, phenobarbitone
induces hepatic glucuronyl transferase activity and may lower
circulating concentrations of unconjugated bilirubin in the
Crigler–Najjar syndrome type 2. Vitamins act as cofactors in
certain enzymatic reactions and can be effective if given in doses
above the usual physiological requirements. For example,
homocystinuria may respond to treatment with vitamin B6,
certain types of methylmalonic aciduria to vitamin B12, and
multiple carboxylase deficiency to biotin. It may also be possible
to stimulate alternate metabolic pathways. For example,
thiamine may permit a switch to pyruvate metabolism by means
of pyruvate dehydrogenase in pyruvate carboxylase deficiency.
inborn errors of the metabolism
In another group of inborn errors of the metabolism the
signs and symptoms are due to deficiency of the end product of
a metabolic reaction, and treatment depends on replacing this
end product. Defects occurring at different stages in
biosynthesis of adrenocortical steroids in the various forms of
congenital adrenal hyperplasia are treated by replacing cortisol,
alone or together with aldosterone in the salt losing form.
Congenital hypothyroidism can similarly be treated with
thyroxine replacement. In some disorders, such as
oculocutaneous albinism in which a deficiency in melanin
production occurs, replacing the end product of the metabolic
pathway is, however, not possible.
signs and symptoms are due to deficiency of the end product of
a metabolic reaction, and treatment depends on replacing this
end product. Defects occurring at different stages in
biosynthesis of adrenocortical steroids in the various forms of
congenital adrenal hyperplasia are treated by replacing cortisol,
alone or together with aldosterone in the salt losing form.
Congenital hypothyroidism can similarly be treated with
thyroxine replacement. In some disorders, such as
oculocutaneous albinism in which a deficiency in melanin
production occurs, replacing the end product of the metabolic
pathway is, however, not possible.
Gene product replacement
Gene product replacement therapy is an effective strategy when
the deficient gene product is a circulatory peptide or protein.
This forms the standard treatment for insulin dependent
diabetes mellitus, haemophilia and growth hormone
deficiency – conditions that can be treated with systemic
injections. This approach is more difficult when the gene
product is needed for metabolism within specific tissues such as
the central nervous system, where the blood–brain barrier
presents an obstacle to systemic replacement.
the deficient gene product is a circulatory peptide or protein.
This forms the standard treatment for insulin dependent
diabetes mellitus, haemophilia and growth hormone
deficiency – conditions that can be treated with systemic
injections. This approach is more difficult when the gene
product is needed for metabolism within specific tissues such as
the central nervous system, where the blood–brain barrier
presents an obstacle to systemic replacement.
Genetically engineered gene products
Genetically engineered gene products are available for
clinical use. Recombinant human insulin first became available
in 1982. The production of human gene products by
recombinant DNA techniques ensures that adequate supplies
are available for clinical use and produces products that may be
less immunogenic than those extracted from animals. In some
cases transgenic animals have been created that produce
human gene products as an alternative to cloning in microbial
systems.
clinical use. Recombinant human insulin first became available
in 1982. The production of human gene products by
recombinant DNA techniques ensures that adequate supplies
are available for clinical use and produces products that may be
less immunogenic than those extracted from animals. In some
cases transgenic animals have been created that produce
human gene products as an alternative to cloning in microbial
systems.
potential problem associated with gene product
A potential problem associated with gene product
replacement is the initiation of an immunological reaction to
the administered protein by the recipient. In haemophilia, the
effectiveness of factor VIII injections is greatly reduced in the
10–20% of patients who develop factor VIII antibodies. The
efficiency of replacement therapy is, however, demonstrated by
the increase in documented life expectancy for haemophiliacs
from 11 years in the early 1900s to 60–70 years in 1980. The
reduction in life expectancy to 49 years between 1981 and 1990
reflects the transmission of the AIDS virus in blood products
during that time period, when 90% of patients requiring
repeated treatment became HIV positive. Factor VIII extracts
are now highly purified and considered to be free of viral
hazard, and recombinant factor VIII has been available
since 1994.
replacement is the initiation of an immunological reaction to
the administered protein by the recipient. In haemophilia, the
effectiveness of factor VIII injections is greatly reduced in the
10–20% of patients who develop factor VIII antibodies. The
efficiency of replacement therapy is, however, demonstrated by
the increase in documented life expectancy for haemophiliacs
from 11 years in the early 1900s to 60–70 years in 1980. The
reduction in life expectancy to 49 years between 1981 and 1990
reflects the transmission of the AIDS virus in blood products
during that time period, when 90% of patients requiring
repeated treatment became HIV positive. Factor VIII extracts
are now highly purified and considered to be free of viral
hazard, and recombinant factor VIII has been available
since 1994.
Cellular Transplantation
An alternative method of replacement is that of organ or
cellular transplantation, which aims at providing a permanent
functioning source of the missing gene product. This approach
has been applied to some inborn errors of metabolism, such as
mucopolysaccharidoses, using bone marrow transplantation
from matched donors. Again, the blood–brain barrier prevents
effective treatment of CNS manifestations of disease. The
potential for direct replacement of missing intracellular
enzymes in treating inborn errors of metabolism is also being
determined experimentally.
cellular transplantation, which aims at providing a permanent
functioning source of the missing gene product. This approach
has been applied to some inborn errors of metabolism, such as
mucopolysaccharidoses, using bone marrow transplantation
from matched donors. Again, the blood–brain barrier prevents
effective treatment of CNS manifestations of disease. The
potential for direct replacement of missing intracellular
enzymes in treating inborn errors of metabolism is also being
determined experimentally.
Gene Therapy
The identification of mutations underlying human diseases has
led to a better understanding of the pathogenesis of these
disorders and an expectation that genetic modification may
play a significant role in future treatment strategies. No such
treatments are currently available, but many gene therapy trials
are underway.
The first clinical trials in humans were initiated in 1990 and
since then over 150 have been approved. Most of these have
involved genetic manipulation in the therapy of cancer, some
have involved infectious diseases or immune system disorders
and a few have involved inherited disorders, notably cystic
fibrosis. Human trials are all aimed at altering the genetic
material and function of somatic cells. Although gene therapy
involving germline cells has been successful in animal studies
(for example curing thalassaemia in mice) manipulation of
human germline cells is not sanctioned because of ethical and
safety concerns. So far, results of human gene therapy trials
have been disappointing in terms of any long-term therapeutic
benefit and many technical obstacles remain to be overcome.
led to a better understanding of the pathogenesis of these
disorders and an expectation that genetic modification may
play a significant role in future treatment strategies. No such
treatments are currently available, but many gene therapy trials
are underway.
The first clinical trials in humans were initiated in 1990 and
since then over 150 have been approved. Most of these have
involved genetic manipulation in the therapy of cancer, some
have involved infectious diseases or immune system disorders
and a few have involved inherited disorders, notably cystic
fibrosis. Human trials are all aimed at altering the genetic
material and function of somatic cells. Although gene therapy
involving germline cells has been successful in animal studies
(for example curing thalassaemia in mice) manipulation of
human germline cells is not sanctioned because of ethical and
safety concerns. So far, results of human gene therapy trials
have been disappointing in terms of any long-term therapeutic
benefit and many technical obstacles remain to be overcome.
classical gene therapy
The classical gene therapy approach is to introduce a
functioning gene into cells in order to produce a protein
product that is missing or defective, or to supply a gene that
has a novel function. This type of gene augmentation approach
could be appropriate for conditions that are due to deficiency
of a particular gene product where the disease process may be
reversed without very high levels of gene expression being
required. Autosomal recessive and X linked recessive disorders
are likely to be the best candidates for this approach since most
are due to loss of function mutations leading to deficient or
defective gene products. Augmentation gene therapy is not
likely to be successful in autosomal dominant disorders, since
affected heterozygotes already produce 50% levels of normal
gene product from their normal allele. In these cases, gene
therapy is not likely to restore gene product production to
levels that will have a therapeutic effect. In neoplastic disorders
the classical gene therapy approach aims to introduce genes
whose products help to kill malignant cells. The genes
introduced may produce products that are toxic, act as
prodrugs to aid killing of cells by conventionally administered
cytotoxic agents, or provoke immune responses against the
neoplastic cells.
functioning gene into cells in order to produce a protein
product that is missing or defective, or to supply a gene that
has a novel function. This type of gene augmentation approach
could be appropriate for conditions that are due to deficiency
of a particular gene product where the disease process may be
reversed without very high levels of gene expression being
required. Autosomal recessive and X linked recessive disorders
are likely to be the best candidates for this approach since most
are due to loss of function mutations leading to deficient or
defective gene products. Augmentation gene therapy is not
likely to be successful in autosomal dominant disorders, since
affected heterozygotes already produce 50% levels of normal
gene product from their normal allele. In these cases, gene
therapy is not likely to restore gene product production to
levels that will have a therapeutic effect. In neoplastic disorders
the classical gene therapy approach aims to introduce genes
whose products help to kill malignant cells. The genes
introduced may produce products that are toxic, act as
prodrugs to aid killing of cells by conventionally administered
cytotoxic agents, or provoke immune responses against the
neoplastic cells.
Genetic manipulation
Genetic manipulation can take place ex vivo or in vivo. In
ex vivo experiments and trials, cells are removed and cultured
before being manipulated and replaced. This approach is
feasible for therapies involving cells such as haemopoetic cells
and skin cells that can be easily cultured and transplanted. In
in vivo methods, the modifying agents are introduced directly
into the individual.
ex vivo experiments and trials, cells are removed and cultured
before being manipulated and replaced. This approach is
feasible for therapies involving cells such as haemopoetic cells
and skin cells that can be easily cultured and transplanted. In
in vivo methods, the modifying agents are introduced directly
into the individual.
augmentation gene therapy
To be effective, augmentation gene therapy requires
methods that ensure the safe, efficient and stable introduction
of genes into human cells. The production of adequate
amounts of gene products in appropriate cells and tissues is
needed with appropriate control of gene expression and
reliable methods of monitoring therapeutic effects. Before
application of gene therapy to humans, in vitro studies are
needed together with proof of efficiency and safety in animal
models. The possibility of insertional mutagenesis and the
dangers of expressing genes in inappropriate tissues need to be
considered. There may also be immunological reactions
mounted against viral vector material or the gene product itself
if this represents a protein that is novel to the individual being
treated.
methods that ensure the safe, efficient and stable introduction
of genes into human cells. The production of adequate
amounts of gene products in appropriate cells and tissues is
needed with appropriate control of gene expression and
reliable methods of monitoring therapeutic effects. Before
application of gene therapy to humans, in vitro studies are
needed together with proof of efficiency and safety in animal
models. The possibility of insertional mutagenesis and the
dangers of expressing genes in inappropriate tissues need to be
considered. There may also be immunological reactions
mounted against viral vector material or the gene product itself
if this represents a protein that is novel to the individual being
treated.
Classical gene augmentation therapy
Classical gene augmentation therapy is not suitable for
disorders that are due to the production of an abnormal
protein that has a harmful effect because of its altered
function. This applies to autosomal dominant disorders where
the mutation has a dominant negative effect, producing a
protein with a new and detrimental function, as in Huntington
disease. Genetic manipulation in this type of disorder requires
targeted correction of the gene mutation or the inhibition of
production of the abnormal protein product. Several
methodologies involving DNA or RNA modification are
currently being investigated.
Other approaches to gene therapy include the increased
expression of protein isoforms not normally expressed in the
affected tissue, or the upregulation of other interacting genes
whose products may ameliorate the disease process. In
Duchenne muscular dystrophy, for example, it is possible that
upregulation of a protein called utrophin, that is related to
dystrophin, may have some beneficial effect in slowing the
progression of muscle damage.
disorders that are due to the production of an abnormal
protein that has a harmful effect because of its altered
function. This applies to autosomal dominant disorders where
the mutation has a dominant negative effect, producing a
protein with a new and detrimental function, as in Huntington
disease. Genetic manipulation in this type of disorder requires
targeted correction of the gene mutation or the inhibition of
production of the abnormal protein product. Several
methodologies involving DNA or RNA modification are
currently being investigated.
Other approaches to gene therapy include the increased
expression of protein isoforms not normally expressed in the
affected tissue, or the upregulation of other interacting genes
whose products may ameliorate the disease process. In
Duchenne muscular dystrophy, for example, it is possible that
upregulation of a protein called utrophin, that is related to
dystrophin, may have some beneficial effect in slowing the
progression of muscle damage.
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