Molecular Basis of Disease Flashcards
Monogenic vs Complex Diseases
Monogenic
- stronger genetic component
- causative gene directly leading to disorder
- recognisable inheritance patterns, hereditary
- gene mutations
- rare
Complex
- more involvement of environment, multifactorial/polygenic
- susceptibility gene increases risk but doesn’t directly cause disorder
- no clear inheritance pattern, familial
- gene variants
- common in population e.g. cancer, CVS disease
Recap basic genetics: chromosomes in our body, DNA bases, alleles, protein synthesis, genotype vs phenotype, epigenetics
22 autosomes and 1 sex chromosome
4 DNA bases: adenine, thymine, cytosine, guanine
Alleles = different DNA sequences at a gene or locus (homozygous vs heterozygous)
Protein synthesis: replication of DNA –> transcription into mRNA –> translation into proteins
- every amino acid coded by 3 consecutive bases
Genotype = sequence Phenotype = anatomical or physiological manifestation
Epigenetics = modifications in gene expression without changing DNA sequence e.g. DNA methylation at CpG islands
Sequence variation: Mutation vs Polymorphism
Mutation = disease-causing change
Polymorphism = changes found in >1% population which are not necessarily disease-causing
Mutations - point mutations (definitions) and trinucleotide repeat expansion
Point mutations:
- Substitution
- silent = no change in amino acid sequence of the protein
- missense = change in codon for ONE amino acid, SIZE of mRNA unchanged but COMPOSITION and possibly FUNCTION of protein does change
- non-sense = change to a STOP CODON e.g. UAA, UAG, UGA with premature termination and truncated protein - Insertion and deletion: frameshift mutations
Trinucleotide repeat expansion:
- error in DNA replication
- dynamic mutations
Structural (chromosomal) variations (3)
Copy number variation
- change in copy number of one particular chromosome region (>50bp)
Chromosomal translocation, deletion and inversions
- translocation = exchange of genetic material between two non-homologous chromosomes
- -> gene may be DISRUPTED OR NEW FUSION GENE
Aneuploidies
- abnormal copy numbers of one or more chromosome
Mendelian inheritance - characteristics of specific inheritance patterns
Strong correlation between disease genotype and disease
Autosomal:
= BOTH GENDERS EQUAL
- dominant = each generation affected; 50% risk of passing mutated allele from parent to child
- recessive = not each generation affected; no known FHx of the disease; consanguinity (25% affected if both parents are carriers)
X-linked:
= NO FATHER-SON transmission (affected male passes allele to all daughters)
- mostly recessive = incidence much higher in male (hemizygous for X-linked genes)
- dominant = both male and females affected
Non-Mendelian inheritance: Mitochondrial inheritance - mechanism, pattern, heteroplasmy, bottleneck, how to confirm mutation, is prenatal diagnosis useful?
Mitochondrial genome (or nuclear genome) mutation
- either maternally inherited or de novo
- pattern of inheritance similar to XLR but only females pass and females are also affected
Heteroplasmy
- co-existence of normal and mutant mtDNA (some functional proteins still present)
- mutation load above a certain threshold = disease
- tissue specific mutation load = variable manifestation in different tissues (liver, brain, heart, kidney generally most affected)
- blood precursor cells has selection against some mutations (die if they carry mutation above load) –> less mutations circulating in blood
Bottleneck effect
- only a small subset of mtDNA molecules is passed from mother to germ cells
- completely random (can be high/low proportion of mutant mtDNA)
- difficult to predict manifestations in child
How to confirm presence of mutation:
- skeletal muscles, urine samples, tissue with high energy demand
Prenatal diagnosis not helpful:
- load of mutation in foetal tissues not correlated with load in other tissues of newborn
- mutant load difficult to predict due to heterplasmy and bottleneck
- mutant load may change with time
Non-Mendelian inheritance: Genomic imprinting
Expression of gene only from one allele –> activity depending on whether the variant was inherited from mother or father (parent-of-origin specific)
- due to transcriptional silencing/epigenetic changes during gametogenesis
- imprinting pattern different in sperm/oocyte
E.g. Prader-Willi syndrome: deletion in paternal homologue
Angelman syndrome: deletion in maternal homologue
Non-Mendelian inheritance: Dynamic mutations - definition/characteristics, examples
PROGRESSIVE EXPANSION of “triplet repeats”
ANTCIPATION = symptoms in later generations often more severe and appear at progressively younger ages
Examples: Fragile X, Huntington, Myotonic dystrophy, Friedrich ataxia, Kennedy disease
Penetrance and Expressivity
Penetrance = % individuals with given genotype who exhibit the associated phenotype
Expressivity = number of symptoms in the presentation of a disease in individuals who have associated phenotypes
Genetic testing: somatic vs germline, de novo vs inherited
Detect mutations
- germline - in gametes –> all cells of offspring –> half of affected individual’s gametes carry mutation
vs
- somatic - occur after fertilisation –> affect certain cell types only –> no gametes carry mutation
- de novo vs inherited
- -> all somatic mutations are de novo
- -> germline mutations can be de novo or inherited
Example: MEN2 - approach to inherited cancer syndrome, implication of genetic testing
Approach to inherited cancer syndromes
- clinical criteria –> molecular analysis –> risk assessment models
MEN2 (2a, 2b and FMTC)
- AD missense “gain of function” mutation of RET oncogene
- high risk of developing MTC –> MEN2 accounts for 25% of MTC
Implications of genetic testing for MEN2 in MTC
- screening of other associated conditions/ tumours
- family members at risk of inheriting MEN2
- ?assess need for prophylactic surgery
Example: MEN2 - inherited cancer syndrome, mutation, implication for genetic testing
Approach to inherited cancer syndromes
- inherited mutation causing increased risk of developing certain tumours at early age
- most are AD, germline mutations
- clinical criteria –> molecular analysis –> risk assessment models (surveillance and prevention)
MEN2 (2a, 2b and FMTC)
- AD missense “gain of function” mutation of RET oncogene
- high risk of developing MTC –> MEN2 accounts for 25% of MTC
Implications for genetic testing for MEN2 in MTC
- screening of other associated conditions/ tumours
- family members at risk of inheriting MEN2
- ?assess need for prophylactic surgery
Example: Wilson’s disease - mutation, stepwise testing approach, implications for genetic testing
AR disorder of copper metabolism
ATP7B mutations with systemic copper accumulation and multi-organ damage
- impaired copper incorporation into apoceruloplasmin
- permanent damage can be prevented if early chelation therapy given
Low serum ceruloplasmin levels has low PPV –> need molecular diagnosis
STEPWISE TESTING APPROACH
- tiered screening approach based on previous studies identifying most common locations of mutant alleles
- start with most common then broaden if negative results
Implications for genetic testing in family members
- overlapping biochemical results between heterozygotes (CARRIERS) and pre-symptomatic (AFFECTED but no organ damage yet) individuals
- early identification and treatment prevents permanent damage
- can use direct mutation analysis since know the mutation points from proband
Example: Fragile X syndrome - mutation, inheritance characteristic
XR disorder
Most common single gene cause of autism spectrum disorders
Transcriptional silencing of FMR1 gene –> CGG REPEATS EXPANSION (trinucleotide repeats) at the 5’ untranslated region
Normal = 5-44 repeats
Pre-mutation (carrier) = 50-200 [normal transmitting male and normal female]
Full mutation = >200 [diseased male, milder phenotype in female]
Trinucleotide repeat in the premutation range is unstable and may EXPAND TO FULL MUTATION AS X CHROMOSOME IS PASSED ONTO NEXT GENERATION
Genetic basis of complex diseases - characteristics
Cumulative and interactive
More than one gene locus involved, susceptibility gene e.g. DM
Each gene contributes modestly to disease
Disease associated with certain POLYMORPHIC VARIANTS of the genes (>1% of population)
Environmental risk involved
e.g. cancer, dementia, CVS disease
DNA polymorphism - definition, types
Regions where more than one allele can occupy the position = polymorphic sites
- reasonably common to see variation within population (GREATER THAN 1%)
- known sites canbe used as marker of genomic location
Types:
- short tandem repeats (2-13 bp)
- **single nucleotide polymorphism (SNP) –> 80% of polymorphisms in human genome
==> analysed in genome wide associated study (GWAS)
- copy number variation (>50bp)
Cancer as a complex genetic trait - main type of mutation, importance of FHx
SOMATIC MUTATION as the basis for development and progression of all types of cancer
- only affects specific types of cells
- cancer associated genes (oncogenes/TSG) with numerous mutations
- can have different genetic components of same cancer e.g. EGFR mutation in lung cancer –> potential therapeutic target
Germline mutations can predispose to the development of somatic mutations for cancer – FHx is important!
Germline mutation + somatic mutation –> multiple tumours, early onset, bilateral
Normal gene + 2x somatic mutations –> single tumours, unilateral, later onset
Genetic testing purposes - reasons for diagnostic/carrier testing, prenatal, preimplantation genetic diagnosis, predictive/presymptomatic, pharmacogenetic testing, genetic screening
Diagnostic and Carrier testing
- diagnostic = symptomatic individual to confirm/exclude
- carrier = mutation detection that generally has limited or no consequence to health
Prenatal diagnosis
- during pregnancy where there is increased risk for a certain condition in the foetus
e. g. chorionic villus sampling/amniocentesis, cicrulating foetal DNA in maternal plasma
Preimplantation genetic diagnosis
- testing the presence of mutation/chromosomeal change in EMBRYO in a family with previously o=known risk for inherited disorders to select unaffected embryos for implantation in IVF
Predictive or presymptomatic testing
- no features of disorder themselves but have family member with genetic disorder
- -> determine risk, predict onset/severity, prophylactic Tx, disease surveillance, planning for Mx, clinical trials
Pharmacogenetic testing
- test for genetic susceptibility for adverse drug reactions or efficacy of drug treatment (not a mutation)
e. g. HLA-B*1502 for carbamazepine SJS
Genetic screening
- target is not high-risk but systematicaly offered to general population or specific group
e. g. newborn screening for IEM
Reporting genetic testing results (classification of variants and conclusions)
Pathogenic (confirmatory) --> treat Likely pathogenic (consistent) --> treat Variant of uncertain significance Likely benign Benign
(gene, genomic coordinates, reference transcript, nucleotide change, protein change, zygosity)
Family tree and pedigrees
KNOW HOW TO INTERPRET/DRAW
Affected = have clinical presentations/symptomatic
Carriers are NOT AFFECTED!
- read question carefully and only use the info given - don’t assume carrier if no info given
Problems of genetic testing
Sensitivity
- meaning of “negative” result -
- > false negatives (low sensitivity) OR
- > if direct mutation approach, can’t rule out presence of other mutations causing disease
- variant of uncertain significance (VUS) –> don’t know whether it explains the genetic disease (need further workup to see if other studies reported mutation or use functional studies to assess for alterations in protein function)
- cost –> direct=cheaper; screening=expensive
Example: benefits of molecular testing in Menkes Disease
XLR disease of copper transport involving ATP7A gene
- confirm diagnosis
- disease course, prognosis and treatment
- information for prenatal diagnosis or PGD
- family screening (for other female carriers)
- clinical trials
Genetic counselling - elements of pre and post-test counselling
Pre-test counselling
- conditions being tested, pattern of inheritance, symptoms
- reproductive options
- nature of test, indications, limitations, possible results
- alternative options and their risks, benefits, limitations
- cost
- possible emotional impacts, the need to inform relatives about results
- follow-up procedures
Post-test counselling
- results
- cope with emotional impact
- recurrence risk
- testing of additional relatives
- course of action, support groups, followup contacts
Genetic testing techniques
Karyotyping
- whole chromosomes (number, size, shape)
Chromosomal microarray analysis (CMA)
- microdeletions and microduplications
- 5-10kb changes can be detected
- for patients with unexplained developmental delay/intellectual disability/autism spectrum
Sequencing
- exact nucleotide components in genome
- Sanger sequencing (single genes)
- NGS (massively parallel; several genes to whole exome/genome)
Special groups for trinucleotide repeat disorders, mitochondrial genome
Example: Huntington disease - dynamic mutation, reasons for declining genetic testing
AD neurodegenerative disorder
Trinucleotide repeat disease
- number of CAG repeats in HTT gene
- anticipation
Declining testing:
- no effective cure
- concerns about discrimination
- cost of testing
- inability to “undo” knowledge once status known
Gene transcription - splicing
Splicing of introns to form mRNA consisting only of exons
- splice at splice donor sites (2 nucleotides after 1st exon) and splice acceptor sites (2 nucleotides before 2nd exon)
DNA Sequencing - materials, procedure
For novel mutations - screening
Materials
- DNA template (denatured into single strand)
- Taq polymerase (elongate complementary DNA)
- Primer
- deoxynucleotides (dNTP)
- dideoxy (ddNTP) – terminates reaction
- DNA denatured by heat
- Primer annealed to template strand to allow addition of dNTP
- Add: template strand, DNA polymerase and nucleotides into reaction (only add one type of ddNTP to each mixture)
- Many fragments generated in each mixture
- Polyacrylamide gel electrophoresis of 4 reagents
- -> smaller fragments move faster,
- -> each band reflective of ddNTP termination point so read off from smallest fragment upwards
Restrict fragment length polymorphism - procedure, interpretation of results
For known mutations - targeted approach
- Digestion by restriction enzymes (cut DNA at specific recognition sites)
- Analyse size of resulting fragments by gel electrophoresis
- probe specific for RFLP DNA
Possible results:
- normal - one cutting point –> 2 fragments
- homozygous mutation - extra cutting point –> 3 fragments
- heterozygous mutation –> 4 fragments
Example: MELAS
Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes
80% pathogenic variant of mitochondrially encoded tRNA leucine 1
Example: SMA
AR
SMN1 gene mutation
SMN2 produces 15% functional protein –> needed to compensate for SMN1 for survival (normally insignificant in healthy but in SMA it is useful for severity assessment)
Homozygous deletion of SMN1 = SMA diagnosis
>3 copies of SMN2 = milder phenotype
