Unit 2 - Molecular and genetic factors in disease
13
Lecture 3 –
Clinical Presentation of Diseases with
molecular defect
1. Inborn errors of metabolism
Inborn errors of metabolism (IEM) are caused by
mutations that disrupt the normal function of a
biochemical pathway. Most IEM are due to autosomal or
X-linked recessive loss-of-function mutations in genes
encoding specific enzymes or enzymatic co-factors.
Knowledge of the biochemical pathway involved means
that specific blocks have predictable consequences,
including deficiency
of the end product and build-up of intermediary
compounds. Many hundreds of different IEM have been
identified and. Most IEM are restricted to paediatric
practice; however, a growing number may now present
during adult life and some of these are discussed below:
Intoxicating IEM
A subgroup of IEM, termed ‘intoxicating IEM’, can present
as a sudden deterioration in a previously well individual.
Such deteriorations are usually precipitated by some form
of stress, such as infection, pregnancy, exercise or changes
in diet. The intoxication is due to the build-up of
accumulation of intermediary compounds, which will vary
according to the pathway involved. For example, in urea
cycle disorders ammonia is the toxic substance
The intoxication is often associated with derangement of
the acid–base balance and, if not recognised and
treated,will often proceed to multi-organ failure, coma
and death.
The diagnosis of these disorders requires specialist
biochemical analysis of blood and/or urine.
Treatment relies on removal of the toxic substance using
haemodialysis or chemical conjugation, and prevention of
further accumulation by restricting intake of the precursors:
as total protein restriction in urea cycle disorders
Mitochondrial disorders
Disorders of energy production are the most common type
of IEM presenting in adult life
The tissues that are most commonly affected in this group
of disorders are those with the highest metabolic energy
requirements, such as muscle, heart, retina and brain.
Therapy in this group of disorders is based on giving
antioxidants as vitamin C and co-factors that can improve
the function of the respiratory chain.
Storage disorders
Storage disorders are most commonly caused by loss-of
function mutations affecting enzymes involved in
lysosomal degradation pathways.
The clinical consequences depend on the specific enzyme
involved. For example:
Niemann–Pick disease type C is caused by autosomal
recessive loss-of function mutations in either the NPC1 or
NPC2 gene.
These results in hepatosplenomegaly, dysphagia, loss of
speech, early dementia, spasticity, An increasing number of
storage disorders are treatable with enzyme replacement
therapy, making awareness and diagnosis more important.
2. Neurological disorders:
Progressive neurological deterioration is one of the most
common presentations.These diseases are mostly
autosomal dominant , examples would be early-onset
familial forms of dementia, Parkinson’s disease ,
Huntington disease The triplet repeat disorders cause an
interesting group of syndromes and have specific features
Huntington disease:
Huntington disease (HD) is triplet repeat disorders. This
condition can present with
a movement disorder or
weight loss or
psychiatric symptoms (depression, psychosis, dementia)
Or with a combination of all three.
The disease is the result of a [CAG] triplet repeat
expansion mutation in the HD gene on chromosome 4.
Since CAG is a codon for glutamine, the mutation
probably leads to gain of function, as deletions of the gene
do not cause HD, expansion of the repeat to above the
normal range results in neurological disease.
The severity of disease and age at onset are related to the
repeat length. In HD, atrophy of the caudate nuclei is
obvious on magnetic resonance imaging (MRI) of the
brain, and in later stages cerebral atrophy is also apparent.
There is currently no therapy that will alter the
progression of the disease, which will often be the cause
of the patient’s death.
Within families there is a tendency for disease severity to
increase and age at onset to fall due to further expansion
of the repeat, a phenomenon known as anticipation. The
mutation is more likely to expand through the male germ
line than through female.
Unit 2 - Molecular and genetic factors in disease
14
3. Connective tissue disorders
Mutations in different types of collagen, fibrillin &elastin.
Make up the majority of connective tissue disorders.
The clinical features of these disorders vary, depending on
the structural function and tissue distribution of the
protein which is mutated.
For example, autosomal dominant loss-of-function
mutations in the gene encoding elastin cause either
supravalvular aortic stenosis, The most commonly
involved systems are:
skin (increased or decreased elasticity, poor wound
healing)
eyes (myopia, lens dislocation)
blood vessels (vascular fragility)
bones (osteoporosis, skeletal dysplasia)
joints (hypermobility, dislocation, arthropathy
)
4. Learning disability, dysmorphism &
malformations
Congenital cognitive impairment (also called mental
handicap or learning disability) affects about 3% of the
population.
There are important ‘environmental & Genetic’ causes of
cognitive impairment, including:
teratogen exposure during pregnancy
(alcohol,anticonvulsants)
Congenital infections (cytomegalovirus, rubella,
toxoplasmosis, syphilis)
premature delivery (intraventricular haemorrhage)
Birth injury (hypoxic encephalopathy).
Genetic disorders that contribute to the etiology of
cognitive impairment are (Chromosome disorders &
dysmorphic syndromes)
Chromosome disorders
Any significant gain or loss of autosomal chromosomal
material (aneuploidy) usually results in learning disability
and other phenotypic abnormalities, Down’s syndrome is
the best known of these disorders, The DNA analysis can
identify causative structural chromosome abnormalities in
10–25% of cases of significant learning disability.
Dysmorphic syndromes
Almost all dysmorphic syndromes are characterized by
the occurrence of cognitive impairment, malformations
and a distinctive facial appearance associated with various
other clinical features.
Making the correct diagnosis is important, as it has
implications on immediate patient management, detection
of future complications and assessment of recurrence risks
in the family.
Clinical examination remains the mainstay of diagnosis
and the patient often needs to be evaluated by a clinician
who specializes in the diagnosis of these syndromes.
The differential diagnosis in dysmorphic syndromes is
often very wide and this has resulted in computer aided
diagnosis becoming an established clinical tool.
The clinical diagnosis may then be confirmed by specific
genetic investigations, as the genetic basis of a wide range
of dysmorphic syndromes has been identified.
5. Familial cancer syndromes
Most cancers are not inherited but occur as the result of an
accumulation of somatic mutation. However some
families are prone to one or more specific types of cancer
affected individuals tend to present with:
Tumors at an early age and are more likely to have
multiple primary foci of carcinogenesis.
Example
Hereditary non-polyposis colorectal cancer
Hereditary non-polyposis colorectal cancer (HNPCC) is
an autosomal dominant disorder with mutations can occur
in several different genes encoding proteins involved in
DNA mismatch repair that presents with early onset
familial colon cancer, particularly affecting the proximal
colon. Other cancers, such as endometrial cancer, are
often observed in affected families.
Familial breast cancer
Familial breast cancer is an autosomal dominant disorder
that is most often due to mutations in genes encoding
either BRCA1 or BRCA2. Both these proteins are involved
in DNA repair. Individuals who carry a BRCA1 or BRCA2
mutation are at high risk of early-onset breast and ovarian
tumours, and require regular screening for both these
conditions. Many affected women will do Prophylactic
bilateral mastectomy and oophorectomy.
Unit 2 - Molecular and genetic factors in disease
15
General principles of diagnosis
Diagnosis can be made by a careful clinical history and
examination and an awareness and knowledge of rare
disease entities. Although DNA-based diagnostic tools are
now widely used, it is important to be aware that not all
diagnostic genetic tests involve analysis of DNA. For
example, a renal ultrasound can detect adult polycystic
kidney disease. By definition, all genetic testing (whether
it is DNA-based or not) has implications both for the
patient and for other members of the family.
1. Clinical history and examination including
constructing a family tree
The family tree—or pedigree—is a three-generation
family history it may reveal important genetic information
of relevance to the presenting complaint, particularly
relating to cancer. A pedigree must include details
from both sides of the family
any history of pregnancy loss or infant death,
consanguinity,
Details of all medical conditions in family members.
dates of birth and
Age at death.
2. Non DNA-based diagnostic tools:
It may sometimes be more economical or convenient to
measure enzyme activity rather than sequencing the
coding region of the genes involved.
Haemoglobinopathy as sickle-cell disease can be
diagnosed by haemoglobin electrophoresis
Immune deficiencies as hypogammaglobulinaemia can
be diagnosed by Ig levels, Complement levels
Inborn errors of metabolism e.g. phenylketonuria can
be diagnosed by Enzyme assays, amino acid levels
Endocrine disease e.g. congenital adrenal hyperplasia by
Hormone levels, enzyme assays
Renal disease can be diagnosed by e.g. polycystic kidney
can be diagnosed by Radiology, renal biopsy
3. DNA-based diagnostic tools
Polymerase chain reaction (PCR) and DNA sequencing:
PCR involves amplification of DNA from small
quantities of starting material. It is the most important
technique in DNA diagnostic analysis. Almost any tissue
can be used to extract DNA for PCR analysis, but most
commonly, a sample of peripheral blood is used. The
ability to determine the exact sequence of a fragment of
DNA amplified by PCR is also of critical importance in
DNA diagnostics.
((
لالطالع: To detect the mutant gene, two primers (lengths of a
single stranded DNA made complementary to part of the
gene to be tested that bind to the 3' and 5' ends of the normal
sequence are designed. By using appropriate DNA polymerases
(enzymes that build up DNA strand based on its complementary
strand) and thermal cycling, the DNA between the primers is
greatly amplified, producing millions of copies of the DNA
between the two primer sites. The amplified normal DNA and
patient's DNA are then digested with a restriction enzyme that
cuts the amplified DNA into pieces of known sizes e.g. the
normal DNA yields three fragments (67 base pairs, 37 base
pairs, and 163 base pairs long); by contrast, the patient's DNA
yields only two products, an abnormal fragment that is 200 base
pairs (instead of two pairs of 37 and 163 b.p.) and a normal
fragment that is 67 base pairs long. These DNA fragments can
determind by gel electrophoresis (by which we can separate
DNA bands or pieces according to their molecular weight) and
then visualized after staining with special stain under ultraviolet
light..
Hybridization:
This is a procedure used in the diagnosis of genetic and
other pathologies as well as in the diagnosis of cancer.
It is based on the fact that the two DNA strands are not
identical but complementary.
The test is performed by adding a synthetic, single
stranded DNA sequence (called a probe) [that is made
complementary to a specific region of DNA under study
and is being labeled with a specific dye] to the double
stranded DNA from the patient (after making it single
stranded by a process called denaturation). If the probe
found its complementary region along the patient's DNA,
it'll combine (hybridize) to it and starts emitting a color or
"fluoresce". This emitted color can be detected using a
UV-microscope.
This procedure forms the basis of what is known as
fluorescent in situ hybridization (FISH).
Nevertheless, this procedure cannot detect single point
mutations or even addition / deletion of 2 or more
nucleotide bases.
So, the technique used for detection of such smaller
defects is usually DNA-based; the most representative and
most commonly used one is polymerase chain reaction
(PCR) that revolutionalized the diagnostic ability of
genetic testing. Most new techniques used nowadays are
PCR-based.