Molecular Basis of Disease
Molecular medicine examines the origin and course of illness and disease and how they can be prevent and treated.
Genetic - inherited failure in one or more components
Behavioral - Exercise Diet Smoking Alcohol Consumption
Environmental - Infections, toxic substances, physical forces
When all 3 factors contribute to disease, the overlap may vary in each case of a disease.
Genetic Disorders -
- Single Gene Disorders -Autosomal Recessive -Autosomal Dominant -Sex-linked
- Multifactorial or complex disorders
- Chromosome Imbalances
- Mitochondrial Disorders
Some monogenic Recessive Disorders
A single gene change causes these recessive disorders
| Disease | Frequence | Symptoms |
|---|---|---|
| Cystic Fibrosis | 1 in 2000 (N. Europeans) | Recurrent Lung infections, pancreatic exocrine deficiency, male sterility.| |
| Phenylketonuria | 1 in 2000 to 1 in 5000 | Mental retardation |
| Tay-Sachs Disease | 1 in 3000 (Ashkenazi jews) | Neurological degeneration, blindness, and paralysis. |
| Thalassaemia | 1-2 in 100 (Mediterranean and Asia in malaria endemic regions) | Anaemia |
| Sickle Cell anaemia | 1-2 in 100 (parts of Africa where marlaria is endemic.) Also in India, the Caribbean, the Middle East, and the Mediterranean | Anaemia |
| Galactosaemia | 1 in ever 55,000 newborns | Kidney failure, an enlarged liver, cataracts (clouding of the eye lens), poor growth, and mental retardation |
Sickle Cell Disease (SCD) -
Autosomal Recessive Disorder
SCD alpha and beta thalasaemies) caused by an inherited structural abnormalit of globin
Affects formation of the protein haemoglobin.
Haemoglobin -
- Two alpha subunits
- Two beta subunits, and
- each subunit complexed with one haem molecule. In vertebrates, haemoglobin tranposrts oxygen from the lungs to the rest of the body.
Sicle Cell Anaemia (SCA)
cause by point (single base) mutation in beta globin gene.
Protein = defined amino acid sequence
mutation changes a single protein (amino acid) building block in beta haemoglobin.
The amino acid glutamic acid (GAG) is replaced with the amino acid valine (GTG) at position 6 in beta-haemoglobin - Glu6Val (Glu is changed to val at position 6)
Pathophysiology of SCA
Sickle cell hemoglobin forms long, inflexible chains
Normal red blood cells are compact and flexible, enabling them to squeeze through small cappilaries
Sickle red blood cells can form blood clots easier.
HbA → Glu replaced by Val in Beta chains → HbS
RBCs → Deoxygenation → Sickled RBCs
Kidney iinfarct → Microvascual occlusion → Necrotic tubuules
Sickle shaped RBCs die prematurely, leading to shortage of RBCs (anaemia).
Sickled RBCs can also block small blood vessels, causing pain and organ damage.
Molecular Diagnostics of SCA
- Target mutation Analysis
- ASO (3 wells, if first lights up it’s disease, second lights up - y, third - z)
- RFLP; mutation can be identified directly in DNA by use of either of 2 restriction endonucleases -Ddel or Mstll. The nucleotide substitution alters a specific cleavage site recognized by each of these 2 enzymes.
- Prenatal Diagnosis
- FISH
- Preimplantation genetic analysis
- PCR, FISH
Identification of SCD using PCR and ASO probes
insert diagram here
Management and Treatment of SCA
Management -
- Prevent tissue deoxygenation
- promote proper hydration
- avoid sources of infection
- prompt medical attention when sick.
Treatment -
- Medication
- Transfusion
- Bone marrow transplantation
- Gene therapy
Inheritance pattern & prevention
Autosomal recessive inheritance
Follow punitts square.
Sickle Cell Disease Corrected in Human Models using Stem Cell-based Gene Therapy.
Combined RNA interference with globin gene transfer to create a therapeutic transgene
The new gene had two functons
- Produce normal haemoglobin and
- suppress the generation of sickle shaped haemoglobin S. The therapeutic gene was engineered into a lentiviral vector and introduced into haematoploietic stem cells to produce normal haemoglobin.
Vectors is just the way you produce these genes and to amplify them
Treatment of SCD with CRISPR/Cas9 Gene Editing Technology
By using CRISPR/Cas9 gene editing technology, it is possible to activate gene expression, gene repression, histone modification, epigenetic alterations, and paint genomic sequences or tag sequence regions with flourescent protein, or protein tag.
Assembly of CRISPR complex - > Associate with target DNA - > Induction of double-strand breaks - > Insertion of donor DNA.
CRISPR isn’t FDA approved because they don’t know yet that the right gene is being replaced or whether it’s accurate enough.
Autosomal Dominant Disorders
One or two dominant alleles myo- muscles -tonia movement
| Disorder | Frequency | Symptoms |
|---|---|---|
| Huntingon’s Disease | 1-2 in 10,000 | Involuntary choreiform movements, dementia, late onsent |
| Myotonic Dystrophy | 1 in 8,500 | Myotonia, heart defects, cataracts |
| Familial hypercholesterolaemia | 1 in 500 | Premature heart disease |
| Familial breast cancer BRCA1 BRCA2 | 1 in 800(USA) 1 in 100(Ashkenazi Jews) | High lifetime risk of breast cancer and ovarian cancer. Earlier onset than sporadic cases |
| Familial Alzeimer’s disease | 10% lifetime risk at age 80 | Dementia, Earlier onset than sporadic cases |
Huntington’s Disease(HD)
Caused by a mutation in HD gene on chromosome 4
The coding region of this gene contains the DNA sequence (CAG) repeated again and again.
The number of times this triplet is repeated varies from person to person ranging from 10 to 26 times. People with HD have an abnormally high number of these CAG triplets, approximately 40 or more.
Premutation range: Individuals are normal but their offspring are at risk of developing the disease. As the altered HD gene is passed from one generation toe the next, the sice of the CAG repeat expansion can change. It often increases tin size especially when it is inherited from the father
The CAR repeat expansion leads to the production of huntington protein that contains an abnormal number of glutamines at the N-terminal This likely disrupts the function ofthe gene’s protein product, hungtintin
The job of its protein product,. huntingtin, is to direct the delivery of small packages (vesicles containt important molecules) to the outside of the cell.
Cellular enzymes cut this elongated protein into fragmments that have “sticky” ends.
the progtein fragments form abnormal clumps inside nerve cells and may attract other, normal proteins into the clumbs
These nerve cells do not function properly
- Neurogenerative disease
Age of Onset versus Number of CAG repeats
The greater the number of CAG repeats the earlier the onset of systptoms
Management and treatment of HD
Medications ease feelings of depression and anxiety, others control involuntary movement
Physical or speechtherapy helps HD patients lead more normal lives
Gene silencing or RNA interference is a promise technique to treat HD.
X-linked Disorders
Affects the genes located on the x-chromosome
Males are much more likely to be affect because they have only one copy of X chromosome.
Can affect male children of unaffected parent.
Females are mostly carriers and are unaffected
Sometimes females may show some symptoms of the disease because of X-chromosome inactivation
| Disorder | Frequency | Symptoms |
|---|---|---|
| Haemophilia A and B | 1 in 10,000 | Abnormally prolonged bleeding after trauma |
| Duchenne Muscular Dystrophy | 1 in 3000 to 1 in 4000 | Muscle wastage in teenage years |
| Fragile-X syndrome | 1 in 1000 | Mental retardation |
| X-linked sever combined immunodeficiency | 1 in 50,000 to 100,000 briths | Recurrent and presistent infections |
Sever Combined Immunodeficiency (X-SCID)
Caused by a mutation in the SCIDX1 gene located on the X chromosome
This gene encodes a protein that is used to contrsuct a receptor called interleukin-2 receptor, subunit gamma(IL2RG) also cytokine receptor common gamma chain.
Cytokine receptor common gamma chain is found on the surface of immune cells (T cells, natural killer, and Beta cells) and allow these cells to communicate.
When the SCIDX1 gene is mutated the IL2RG receptor cannot form properly or are absent from immune cells.
As a result, the immune cells can’t communicate with one another about invaders in the environment.
Insufficient T and Beta cells are produced to fight off the infection, and the body is left defenseless.
Mutation leading to X-SCID
Mutation in IL2RG-receptor gene may be cause by:
- Missense - both are types of point mutation (single gene change)
- Nonsense - ^
- Insertion - When a gene is removed and replaced
- Deletion mutations and, - When a gene is just removed
- Splice defects - When they start splicing
Molecular Diagnostic testing
- Sequence analysis. Sequence analysis of the IL2RG coding region.
- Target Mutation Analysis. Southern blot analysis for the detection of large deletions and complex mutations for individuals in whom mutations are not detected by sequence analysis.
Complexity of Single-gene Disorders
A number of factors modify the pattern of inheritance or symptoms of the disorder:
Genetic Heterogeneity:
- Allelic heterogeneity: different mutations within a single gene locus (i.e. multiple alleles of a gene) result in same phenotypic expression. E.g over 1000 known mutant alleles of the CFTR gene cause cystic fibrosis.
- Locus heterogeneity: variations in completely unrelated gene loci cause a single disorder. E.g retinis pigmentosa can have autosomal dominant, autosomal recessive or X-linked origin.
Penetrance: the frequency with which a particular genotype manifests itself in the phenotype - complete or incomplete.
Expressivity: same disease alleles but differences in severity. (Liked huntingtons, normal, premutation, etc.)
Mosaicisms: all body cells are not genetically identical
Phenocopy: Same symptoms (e.g. deafness) may be caused by an enviornmental facotrs (e.g rubella virus infection) or by an inherited disorder).
Environmental effects: e.g. phenylketonuria
Anticipation: severity of disease increases with succeeding gneerations, e.g. muscular dystrophy
Genomic imprinting: expression of disease depends on the parent from which it was inherited. E.g deletion of some genes from chromose 15: father: prader-willi syndrome, mother: angelman syndrome.
Multifactoral or Complex disorders
People with a condition that is due to interaction of genetic and environmental factors, health, growth, development. (Venn diagram between our genes and our physical and chemical environment).
Diseased caused by combined effects of multiple genetic and environmental (age, gender, infection, or nutrition) risk factors
There is no dominance or recessivity at each of these genes
The genes act in cocnert in an aadditive fashion. Each adding or detracting a small amount from the phenotype.
Most risk factors probably of small effect individual and with complex interaction.
The environment interacts wit hthe genotype to produce the final phenotype.
Members of the same family are likely to suffer from the same disease -The are likely to share the same environment They are likely to share similar genes
Examples of Complex Disorders
| Congenital Disorder | Common diseases of later life | Physchiatric Disorders |
|---|---|---|
| Neural Tube - Spinal Bifida - Anencephaly | Rheumatoid arthritis | Manic Depression |
| Congeinital Heart disease | Various Cancers | Alcoholism |
| Cleft lip palate | Epilepsy | Schizophrenia |
| Mental Retardation | Multiple sclerosis | Tourette’s syndrome |
| Insulin-dependent diabetes mellitus | Dyslexia | |
| Non-insulin dependent DM | Alzeimer’s Disease | |
| Peptic ulcer | ||
| Ischaemic heart disease | ||
| Hyperthyroidism | ||
| Migraine | ||
| Asthma |
Multifactoral or Complex disorders
Affect children may have normal parents
Recurrence risk increases with the number of affected children in a family.
A more severely affected parent is more likely to produce an affected child.
Consanguinity slightly increases the risk of an affected child.
Chromosomal Mutations
Entire chromosomes, or large segments of them, are missing, diuplicated, or altered.
Trisomy
Monosomy
Translocation; chromosomes break and the fragments rejoin to other chromosomes.
| Condition | Frequency |
|---|---|
| Sex Chromosomes: | - |
| 45x: Turner’s Syndrome | 1 in 5000 |
| 47XXY: Klinefelter’s syndrome | 1 in 1000 |
| Autosomes | - |
| Trisony 21: Down’s Syndrome | 1 in 800 (maternal age dependent) |
| Trisomy 18: Edward’s Syndrome | 1 in 10,000 |
Mitochondrial Mutations
The number of mitochondria in a cell depend on the metabolic requirements of the cell.
Each mitochondrian ahs 2-10 copies of a 16.6-kb circular DNA genome (mtDNA).
90% of cellular energy is generated in the mitochondria by the respiratory chain.
Mitochondrial diseases comrpsie those disorders tha in one way or another affect the function of the mitochondria and/or are due to mitochondrial DNA mutations.
Mutations in the mtDNA cause a number of the disorders where there is decline in energy availability and other clinical abnormalities.
Mitochondria are maternally inherited, therefore mitochondrial disorders can only be inherited from an individual’s mother.
Different factors lead to great complexity in the age of onsent and severty of symptoms.
Mitochondrial disorders affect many different organs simultaneously include the central nervous system.
Some diseases resulting from mitochondrial mutations
| Disease | Acronym | Symptoms | Affected Gene |
|---|---|---|---|
| Chronic progressive external Opthalomoplegia | CPEO | ||
| Pearson’s Syndrome | |||
| Neurogeinc muscle weakness, ataxia and retinis pigmentosa | NARP | ||
| Many disease are believed to be in part of dysfunction of mitochondira such as |
- Diabetes mellitus
- some forms of cancer
- cardiovascualar disease
- lactic acidosis
- specific forms of myopathy
- osteoporosis
Misc.
Gene ATAGAA
Rhybosome reads in sets of 3s {ATA} {GAA} {etc.} Creates amino acids based on these sets. Amino acids combine to make proteins. Changed a Gene changes the amino acids produced