Molecular Basis of Disease

Question 1

Monogenic vs Complex Diseases

Answer 1

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

Question 2

Recap basic genetics: chromosomes in our body, DNA bases, alleles, protein synthesis, genotype vs phenotype, epigenetics

Answer 2

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

Question 3

Sequence variation: Mutation vs Polymorphism

Answer 3

Mutation = disease-causing change

Polymorphism = changes found in >1% population which are not necessarily disease-causing

Question 4

Mutations - point mutations (definitions) and trinucleotide repeat expansion

Answer 4

Point mutations:

1. 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
2. Insertion and deletion: frameshift mutations

Trinucleotide repeat expansion:

• error in DNA replication
• dynamic mutations

Question 5

Structural (chromosomal) variations (3)

Answer 5

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

Question 6

Mendelian inheritance - characteristics of specific inheritance patterns

Answer 6

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

Question 7

Non-Mendelian inheritance: Mitochondrial inheritance - mechanism, pattern, heteroplasmy, bottleneck, how to confirm mutation, is prenatal diagnosis useful?

Answer 7

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

Question 8

Non-Mendelian inheritance: Genomic imprinting

Answer 8

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

Question 9

Non-Mendelian inheritance: Dynamic mutations - definition/characteristics, examples

Answer 9

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

Question 10

Penetrance and Expressivity

Answer 10

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

Question 11

Genetic testing: somatic vs germline, de novo vs inherited

Answer 11

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

Question 12

Example: MEN2 - approach to inherited cancer syndrome, implication of genetic testing

Answer 12

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

Question 13

Example: MEN2 - inherited cancer syndrome, mutation, implication for genetic testing

Answer 13

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

Question 14

Example: Wilson’s disease - mutation, stepwise testing approach, implications for genetic testing

Answer 14

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

Question 15

Example: Fragile X syndrome - mutation, inheritance characteristic

Answer 15

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

Question 16

Genetic basis of complex diseases - characteristics

Answer 16

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

Question 17

DNA polymorphism - definition, types

Answer 17

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)

Question 18

Cancer as a complex genetic trait - main type of mutation, importance of FHx

Answer 18

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

Question 19

Genetic testing purposes - reasons for diagnostic/carrier testing, prenatal, preimplantation genetic diagnosis, predictive/presymptomatic, pharmacogenetic testing, genetic screening

Answer 19

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

Question 20

Reporting genetic testing results (classification of variants and conclusions)

Answer 20

Pathogenic (confirmatory) --> treat
Likely pathogenic (consistent) --> treat
Variant of uncertain significance
Likely benign
Benign

(gene, genomic coordinates, reference transcript, nucleotide change, protein change, zygosity)

Question 21

Family tree and pedigrees

Answer 21

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

Question 22

Problems of genetic testing

Answer 22

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

Question 23

Example: benefits of molecular testing in Menkes Disease

Answer 23

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

Question 24

Genetic counselling - elements of pre and post-test counselling

Answer 24

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

Question 25

Genetic testing techniques

Answer 25

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

Question 26

Example: Huntington disease - dynamic mutation, reasons for declining genetic testing

Answer 26

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

Question 27

Gene transcription - splicing

Answer 27

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)

Question 28

DNA Sequencing - materials, procedure

Answer 28

For novel mutations - screening

Materials

• DNA template (denatured into single strand)
• Taq polymerase (elongate complementary DNA)
• Primer
• deoxynucleotides (dNTP)
• dideoxy (ddNTP) – terminates reaction

1. DNA denatured by heat
2. Primer annealed to template strand to allow addition of dNTP
3. Add: template strand, DNA polymerase and nucleotides into reaction (only add one type of ddNTP to each mixture)
4. Many fragments generated in each mixture
5. Polyacrylamide gel electrophoresis of 4 reagents
   - -> smaller fragments move faster,
   - -> each band reflective of ddNTP termination point so read off from smallest fragment upwards

Question 29

Restrict fragment length polymorphism - procedure, interpretation of results

Answer 29

For known mutations - targeted approach

1. Digestion by restriction enzymes (cut DNA at specific recognition sites)
2. 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

Question 30

Example: MELAS

Answer 30

Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes

80% pathogenic variant of mitochondrially encoded tRNA leucine 1

Question 31

Example: SMA

Answer 31

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