Session 3: Modes of inheritance and constitutional genetics Flashcards
what are antisense oligonucleotides?
ssDNA or RNA 20bp that binds to mRNA blocking the translational mechanism. The can be used to block production of abnormal protein, correct aberration by exon skipping or correct splice mutations. they are checmically modified to prevent nuclease degradation
give examples of how antisense oligos have been used to treat diseases?
- DMD - Eteplirsen (Exondys 51) in clinical use. used as reading frame correction to induce exon skipping and produce smaller partially functional protein. requires repeated treatments. it is mutation specific but due to hotspots is applicable to wide number of patients. low efficiency in heart muscle and many patients die of heart complications so need to improve AO efficiency in heart muscles - would allow lower dosage and fewer administrations
- SMA - Nusinersen (Spinraza) in clinical use enhance exon 7 inclusion in SMN2 to produce functional protein and lessen SMA caused by loss of SMN1. this results from blocking intron 7 splice site to promote exon 7 inclusion.
- HD - IONIS-HTTRx in phase 3 trials - suppress translation of HTT mRNA containing CAG expansion. targets HTT-dependent SNPs so doesnt taget expansions in other genes.
what are the Challenges of using ASOs for the treatment of genetic diseases?
o Delivery to target tissue
o Achieve sustained effect. Chemically stabilised forms of ASOs will require re-administration for most applications.
o Difficult to achieve complete inhibition as there are large quantities of mRNA and lower levels of ASO within the cell.
describe CRISPR-Cas9 system and limitations
CRISPR-Cas9 is a highly cost-effective technique that allows specific targeting of gene manipulation via RNA-guided nuclease. once a cut is made, DNA with matching sticky ends can be incorporated by DNA repair mechanisms. Trials in b-thalassaemia, DMD, an
d freidreich ataxia.
limitations:
1. limited number of motifs to bind to in genome
2. delivery to target cells
3. although it has proofreading finction, some off target mutagenesis has been seen in similar sequences to target
describe small molecules for genetic treatment
- amnioglycosides promotes read-through of stop codons. amnioglycosides interact with A site of rRNA, altering conformation and reducing accuracy between mRNA-tRNA pairings allowing AA to be inserted instead of termination at stop codon. works best where low levels of functional protein can restore function. eg. Translana in DMD induces ribosomes to read through PTC. works on mRNA transcripts so patients with low mRNA levels may not respond. NICE approval
- corrects folding/transport or activation of protein eg. Ivacaftor for CFTR to improve chloride channel transport (class III) or enhance folding (class II) eg. phe508del Lumacaftor.
what are features of x-linked recessive inheritance?
- vertical transmission of carrier females to affected sons (50% chance)
- all daughters of affected males have the pathogenic variant (obligate carriers)
- daughters of female carriers have 50% chance of being a carrier
- affected homozygous females are rare
- affected males usually born to unaffected parents (may have affected male relatives)
- no male to male transmission
- pedigree mostly affected males, females are carriers only
why might females be affected by XLR disorder?
- skewed inactivation
- X chr deletion
- x chromo translocation
- variable penetrance/expressivity
- XO
- uPD
- compound heterozygosity
Give examples of X linked disorders?
- DMD, BMD,
-SBMA - androgen insensitivity syndrome
- XLRP (retinitis pigmentosa)
- haemophilia A and B - Christmas disease
what is DMD?
- 1/4000 births affected
- rapidly progressive muscular weakness proximal > distal & lower> upper limbs with calf hypertophy
- gower movement
- early childhood age of onset & first signs are delayed milestones, delayed sitting and standing
- cardiomyopathy >18 years, most common cause of death with respiratory complications
- wheelchair bound by 12
- few survive beyond 30 yrs
- males do not usually reproduce
- full penetrance in males, females can be unaffected to severe (manifesting carrier)
- > 10x creatine kinase levels, less in females. CK is an enzyme released from muscle into the bloodstream following damage
- out of frame deletions (be careful with duplications as dont always follow the rule)
what is BMD?
-1/18 000 male births
- in-frame deletions in DMD
- less severe & later onset
- longer life expectancy (mid 40s)
wheelchair bound >16
- heart failure from DCM still common cause of death
- females with DMD pathogenic variant at increased risk for DCM
- preservation of neck flexor muscle strength (differs from DMD)
- >5x creatine kinase levels , less in females
what is DMD-associated dilated cardiomyopathy (DCM) ?
- DCM between 20-40 years in males and later in females
- usually no skeletal muscle disease
- rapid progression to death in males and slower progression in females
-increased creatine kinase levels , less in females
how do you test for DMD without genetics?
immuno staining shows lack of dystophin in skeletal muscle, cardiac and smooth muscle cells. Dystrophin plays a role in sarcolemma stability. In DMD it is absent but in BMD it is 20-100% - may be normal levels but reduced function
how do you genetically test for DMD?
- MLPA (80% mutations) - QF-PCR required or single del/dup or SNP array can offer higher resolution may be done solely by NGS in future
- sequencing, NGS allows mosaics to be detected. sanger for familial mutations
- RNA analysis deep intronic variants or complex rearrangements. usually muscle or urine but need to confirm in genomic DNA. can also confirm splicing outcomes and orientation of duplications
Two deletion hotspots: 30% exons 2-19 and 70% exons 45-55
What is traditional treatments for D/BMD (before gene therapies)?
steroids (improve muscle strength and motor function), physical therapy, anti-congestives and cardiac transplant in severe cases
what gene therapy is available for D/BMD?
- stop codon readthrough eg. translana. 15% of patients have PTC. Tretments allows alternative amino acids to be inserted at the site of the mutated stop codon & results in dystophin expression at 10-20% providing some function
- exon skipping eg. exondys51 (80% of patients in theory). Interferes with splicing skipping the specific exon in DMD pre-mRNA to restore open reading frame and allow expression of shorter but functional protein
what is SBMA?
X linked disorder affecting males 1/300 000
caused by CAG repeat in exon 1 of AR gene
progressive neuromuscular disorder with degeneration of motor neurons resulting in muscle weakness and muscle atrophy and reduced fertility (due to mild androgen insensitivity)
GOF mutation. the more repeats there are the earlier the age of onset
only occurs in Europeans or Asians
females not usually affected
what is androgen insensitivity syndrome (AIS)?
complete = total insensitivity to androgen and child develops female genitals. abnormal secondary sexual development at puberty and infertility in those with a 46, XY karyotype
partial = level of insensitivity determines how genitals develop (predominantly female, male or ambiguous)
mild = typically male genitalia
Affects 2-5/100 000 who are genetically male
infertility
What causes androgen insensitivity syndrome?
- pathogenic sequence variants in AR gene (XLR)
- androgen receptors allow cells to respond to androgens (hormones such as testosterone) that direct male sexual development
- AR mutations present receptors from working properly and makes cells less responsive to androgens
WHat is haemophilia A and B (christmas disease)?
XLR disorders caused by mutations in F8 (A) and F9 (B) causing F8 and F9 to be inefficient at coagulation in the blood. Disorders are indistinguishable clinically. Clotting deficiency results in prolongued bleeding after injury. 10% of female carriers are at risk for bleeding. 1/6000 males have haem A and 1/30 000 males have haem B.
what is Fabry disease?
XLR lysosomal-storage disorder caused by mutations in GLA which encodes the galactosidase enzyme (breaks down a fatty substance). pathogenic variants prevent the enzyme from breaking down this substance leading to it damaging cells. symptoms include pain in hands and feet, inability to sweat, cloudiness in eye, angiokeratomas *dark red spots on skin), GI problems, tinnitus and hearing loss. life-threatening complications include kidney damage, heart attack and stroke. 1/5-10 000 affected. childhood onset. females may be affected
define anticipation?
give examples
trinucleotide repeat expansions in successive generations in which the signs and symptoms of some genetic conditions tend to become more severe, more frequent or occur at an earlier age
- these dynamic expansions are unstable and expand on transmission to next generation
- eg. FRAX (XL) maternal CGG expansion
- HD HTT CAG AD paternal
- DM1 CTG DMPK maternal expansion
what causes trinucleotide repeats to expand?
replication slippage - mispairing, hairpin loops causing replication fork blockage, unequal crossing overresulting in one expanded and one contracted tract
define Age-related mosaicism?
Mosaicism is defined as the presence in an individual, or in a tissue, of two or more cell lines that differ in genetic constitution. As we age, somatic/germline mutations accumulate over the course of a person’s life resulting in age-related mosaicism
eg. loss of X in males and females is normal age-related anaphase lag
eg. cancer is an example of age related mosaicism - mutations accumulate as we age
define Variable expressivity
give examples
degree to which phenotype is expressed varies between individuals with same genotype
eg. MArfan FBN1 - some are just tall and thin whilst others have heart conditions
eg. NF1 - mildly affeected have cade au lait skin but more severely affected have neurofibromas. may also develop tumours. may also have ID, short stature or seizures.