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.
define penetrance
give examples
proportion of individuals carrying a particular variant of a gene (allele or genotype) that also expresses an associated trait (phenotype)
eg. BRCA1 - penetrance = 80% risk of BC. also affected by environmental modifiers such as smoking, diet, pregnancies etc
what is Age-related penetrance
penetrance is often expressed as a frequency, determined cumulatively, at different ages
define Sex-limiting
give an example
Sex-limited genes are genes which are present in both sexes but expressed only in one sex, and causes the two sexes to show different traits or phenotypes
eg. Familial Male Precocious Puberty - males only have signs of puberty at 4 years
define epistasis
give an example
interaction between nonallelic genes in which one combination of such genes has a dominant effect over other combinations eg. Bombay phenotype
define pleiotrophy
give an example
Pleiotropy occurs when one gene influences two or more seemingly unrelated phenotypic traits eg. usually metabolic pathway that affects different phenotypes such as PKU. depending on mutation involved may have reduced conversion of phenylalanine to tyrosine or ceased entirely. the failure to convert normal levels of phenylalanine to tyrosine results in less pigmentation being produced causing the fair hair and skin typically associated with phenylketonuria. also causes mental retardation and abnormal gait and posture.
what is antagonistic pleitropy?
the expression of a gene resulting in competing effects, some beneficial but others detrimental to the organism.
Antagonistic pleiotropy hypothesis – some genes responsible for increased fitness in the younger, fertile organism contribute to decreased fitness later in life. An example is the p53 gene, which suppresses cancer, but also suppresses stem cells, which replenish worn-out tissue.
define: Amorphic, Hypomorphic Hypermorphic Antimorph and Neomorphic
Amorphic = LOF
hypomorphic = partial loss through reduced function. usually recessive but occasionally dominant due to haploinsufficiency eg. Friedreich’s ataxia homozygous GAA repeat expansion. some cases are compound het with a point mutation. Homozygosity for inactivating mutations is embryonic lethal.
hypermorphic = GOF - increased activity of normal function
antimorph = dominant negative (gene product adversely affects the normal, wild-type gene product)
NEOMORPHIC - DOMINANT GAIN OF GENE FUNCTION that is different from normal function
what is a transition substitution?
a pyrimidine for a pyrimidine (C for T or vice versa) or a purine for a purine (A for G or vice versa)—
what is a transversion substitution?
Substitution of a pyrimidine by a purine or vice versa (C to an A)
define haploinsufficiency
A situation in which half amount of a gene product is not enough to maintain normal function for instance, individual with heterozygous mutation or hemizygous at a particular locus is clinically affected.
define linkage disequilibrium
give a CF example
: Linkage disequilibrium is the phenomenon by which there is a non-random association of alleles at two or more loci i.e. when variants co-occur together in an allele more than should be expected if random distribution of variants was occurring.
Phe508del 98% occurs in cis with 9T - important if patient also has c.350G>A p.(Arg117His) and 5T (causes CF with 5T only)
give an example of XLD disorder?
Fragile X, - CGG trinucleotide repeat in FMR1
X linked Alport syndrome - COL4A5. most common. kidney disease, hearing loss and eye abnormalities
give an example of an X-linked dominant disorder with male lethality
rett syndrome
Intercontinentia pigmenti
what are the features of XLD inheritance?
- expressed in males and females
- an affected female has a 50% chance of having an affected child
- an affected male has affected daughters and unaffected sons
- higher proportion of females affected than males
- affected males have more severe phenotype - variability in females
- may be mistaken for AD, unless there is an affected male who will have all affected daughters and no affected sons
When the mother is affected with an X-linked lethal disease what is the expected offspring ratio of M:F affected vs unaffected?
1/3 affected females, 1/3 unaffected females and 1/3 affected males
what are the features of an X-linked dominant disorder with male lethality in pedigrees?
- disorder observed exclusively in females
- affected males rarely seen (except XXY)
- history of miscarriages as 50% of males die
- sex ratio of offspring is therefore skewed
describe X-linked male lethal Intercontinentia pigmenti
- affects hair, skin, nails. teeth eyes and CNS
- wart-like skin growths
- Majority of males spontaneously abort
- NEMO gene with common deletion
- without NEMO protein, cells triggered to self-destruct
- high penetrance and highly variable
describe X-linked male lethal rett?
- neurological and developmental disorder with repetitive, stereotypic hand movements
- mecp2
- de novo
- high variability in females
- males with XXY or somatic mosaics survive
why is XLD Craniofrontonasal syndrome caused by variants in EFNB1 more severe in females?
-Cellular ‘interference’ between wild-type and mutant cell populations is the cause for the severe disease manifestation in CFNS females
-males are asymptomatic/mild phenotype
why might an XL disorder present in a female?
- skewed x inactivation
- variants in PAR regions are expressed on both X’s as escape X inactivation and can behave in AD manner eg. SHOX on PAR1
- XLD
- XO
- deletion on one X
ADD TO CARDS what is a microdeletion? give examples of reciprocal, interstitial and terminal microdeletion syndromes.
<5-10 Mb, can be recurrent
-monosomy and haploinsufficiency of dosage sensitive –genes. Also involves imprinted genes, unmasking recessive mutation, and positional effects
- can result in clinically recognisable syndrome
can be mendelian or contiguous eg. di george syndrome
for every deletion there should be a duplication - reciprocal microdeletion/duplications from NAHR eg. HNPP/CMT1A 17p12 and Smith-Magenis syndrome (SMS)/Potocki-Lupski syndrome (PTLS) (17p11.2)
Interstitial microdeletions:
Di GEorge 22q11.2
PWAS 15q11-q13
NF1 17q11.2
Miller-Dieker 17p13.3
williams syndrome 7q11.23
terminal microdeletions: wold-hirschorn del4pter
cri du chat del 5pter
what is a microduplication
<5-10 Mb, can be recurrent
regional trisomy and effects dosage sensitive genes
eg. mendelian -PMP22 HNPP,
- milder than deletions as cells more tolerant to gain than loss
- FISH less sensitive for duplications
-variable expressivity and reduced penetrance
Interstitial eg. Duplication 7q11.23 (williams syndrome region duplication syndrome)
what causes microdeletions/microduplications?
- low copy repeats up to 0.5Mb that have high homology >97% and result in structural aberrations from NAHR. NAHR hotspots cause recurrent CNVs
- may be interchromosomal
- intrachromosomal (between 2 chromatids)
- intrachromatid (within chromatid)
- results in deletions, duplications, inversions and dicentric
what methods can be used to identify microdeletions and duplications?
- FISH
- arrayCGH
- MLPA
- Q-PCR
- Optical Genome Mapping (Next gen cyto)
where is Charcot-Marie-Tooth (CMT1A)/Hereditary neuropathy with liability to pressure palsies (HNPP) located and what causes the syndromes? what are clinical features of the syndromes?
17p11.2
- reciprocal 1.4Mb duplication/deletion including PMP22- caused by NAHR between two LCRs with 99% homology
CMT1A (80% of CMT1) = PMP22 duplications. CMT1 is a demyelinating peripheral neuropathy. distal muscle weakness and atrophy, slow nerve conduction. slowly progressive. foot drop, calf hypertrophy.
HNPP - milder neuropathy. numbness, tingling and muscle weakness in the limbs. deletion of PMP22 (80%). other 20% have pathogenic mutation.
ADD TO CARDS where is smith magenis/Potocki-Lupski syndrome located and what causes the syndromes? what are clinical features of the syndromes?
17p11.2
3.7Mb recurrent del/dup generated by NAHR between LCRs. common del/dup occurs due to NAHR between proximal and distal low copy repeats.
smith-magenis syndrome = developmental delay, hypotonia and distinctive facial features. caused by de novo 17p11.2 deletions. (70%)
potocki-lupski syndrome - dev delay, autism and hypotonia. de novo duplications of 17p11.2
what causes Prader-Willi syndrome (PWS)/Angelman syndrome (AS) del 15q11-q13 and what are clinical features?
PWS = hypotonia, feeding difficulties, obesity, dev delay, insatiable appetite.
- paternal contiguous gene deletion resulting from NAHR between low copy repeats (75%) of 15q11q13 leading to absence of expression of paternally-imprinted genes. also caused by mat UPD and IC deletion. there are 4 large clusters of complex repeats termed BP1-4, with common deletion ranging from BP1 or 2 to BP3. SNURF-SNRPN, MAGEL2, C15orf2 involved.
angelmann = severe developmental delay/intellectual disability, severe speech impairment, gait ataxia , happy demeanor, microcephaly, sezures. Disruption of UBE3A in 15q11q13. 70% have maternal deletion.
what causes Neurofibromatosis type 1 (NF1)? where is the location? what are clinical features?
del 17q11.2
multiple café-au-lait spots, neurofibromata, learning difficulties
heterozygous loss of function mutations in the NF1 gene at 17q11.2 as well as by an LCR-mediated 1.5Mb deletion that encompasses NF1 and other flanking genes and pseudogenes
what is the location of Wolf-Hirschhorn syndrome , what causes the syndrome and what are the clinical features?
del 4pter
craniofacial features (greek warrior helmet appearance
dev delay, growth delay, ID, seizures
contiguous gene deletion
up tp 60% de novo and 40% have unbalanced translocation with deletion of 4p and partial trisomy of different chromosome arm.
may require FISH for detection
Candidate genes that may contribute to the WHS phenotype include WHSC1, LETM1 (seizures) and FGFRL1 (craniofacial phenotype).
what is the location of cri du chat syndrome , what causes the syndrome and what are the clinical features?
del 5pter
cat-like cry
severe dev delay/ID
microcephaly, hypotonia, hyperterlorism,
variable seized deletions of 5p from 5p13-5p15
size of del correlates with phenotype
85% are de novo deletion and 15% are unbalanced translocation
CTNND2 causes ID
what is the SMA carrier frequency in uk?
1/50
what is the main clinical symptom of SMA?
- progressive proximal muscle weakness due to spinal cord and motor neuron degeneration
- earlier onset is more severe
- prenatally: Arthrogryposis (joint contractures), absence of movement, death from respiratory failure
describe the different SMA types? what is the most likely cause?
- type 1 = most common form <6 months
- floppy baby
death at early age - poor head control and unable to sit
- swallowing and feeding difficulties
- homozygous deletion
type 2 = <1 year
- low muscle tone
- may be able to sit, never able to walk
- 70% reach adulthood
- gene conversion in one allele and hemi deletion in other
- type 3 = >1 until adulthood
- can stand and walkprobability of being ambulatory decreases with age
- gene conversion in both alleles
type 4 = adult onset
normal life expectancy
<1% of patients
aytypical - non chromosome 5 related
floppiness, multiple fractures, high CK
if a baby presents as floppy, what is the testing triad?
SMA, myotonic dystrophy and PWS
new TD = R70 (separate referral from hypotonic infants which go for R69 DM1, PWS and microarray so now the consultant has to specify SMA)
what causes SMA?
homozygous or compound het deletions and mutations of SMN1
- 98% homozygous deletion of SMN1 exon 7 (deletions or gene conversions)
- 2% compound het for a deletion and mutation
- <1% have homozygous pathogenic mutation in SMN1
mutations may be deep intronic.
2% of deletions are de novo (unequal crossing over)
Incidence of SMA remains high due to high new mutation rate
- germline and somatic mosaicism have been described
why is the SMA gene difficult to test
- has SMN2 (centromeric) pseudogene
- complex region of high instability with repetitive sequences, retrotransposable elements, deletions and inverted duplications
- 4% of population have two copies of SMN1 on one chromosome (can have up to 5 copies)
- SMN1 and 2 differ by 5 base pairs
- critical difference is a synonymous variant in SMN2 exon 7 which alters ESE and 90% of transcripts lack exon 7 in SMN2 and produce truncated product and so is degraded
- SMN2 still produces some full length SMN protein (10%) but not sufficient to compensate for lack of SMN1
- homozygous SMN1 del patients have at least one copy of SMN2 as SMN null mice is embryonic lethal
- higher copies of SMN2 leads to milder phenotypes
what is the SMN protein a component of?
spliceosome
how do you test for SMA?
-98% are deletions so MLPA or real-time PCR for SMN1 exon 7 & 8 deletion
- MLPA uses probes specific to the SNV differences between SMN1 and SMN2
- multiplex real time PCR uses TaqMan probes specific to exon 7 SMN1 and a control probe
- 2% sequence variant - NGS, allele specific long range PCR and RT-PCR/cDNA sequencing are used for specific analysis of SMN1
why might a parent of a child with SMA have normal SMN1 dosage? how can this be confirmed?
- 4% of population have two copies of SMN1 on one chromosome - linkage analysis
- de novo deletion - 2% of patients, can do bayes calculation for normal dosage parent.
what therapies are available for SMA?
what are issues with these treatments?
drugs work by stabilising the SMN protein or modulating SMN2 expression
- Increasing protein:
Zolgensma - virus vector delivers SMN1 transgene to affected motor neurons
modulate SMN2:
Nusinersen/Spinraza is an antisense oligo to promote inclusion of exon 7 in SMN2 transcripts by blocking intron 7 splice site to increase SMN protein levels. highly stable and not toxic.
Risdiplam - oral (not injected)promotes inclusion of exon 7 in SMN2
Issues - usually diagnosedin advanced stages where motor degeneration has occured. screened for?
current pilot study to include SMA in UK newborn screening - March 2022
define haploinsufficiency?
both copies of a gene need to be functional in order to express the wildtype. 50% of the normal gene product/expression/activity is not enough. Inactivation/loss of a single allele (leaving the second allele unaffected) produces a clinical phenotype
A gene is likely to be haploinsufficient if all mutation types (missense, nonsense, gene deletion etc.) produce the same phenotype
give an example of a haploinsufficient single gene?
PMP22 - HNPP 80% caused by deletion of a ~1.5Mb region at 17p11.2 and 20% caused by mutation
repeated focal pressure neuropathies
HNPP is haploinsufficient whilst PMP22 dup CMT1A is GOF
give an example of a haploinsufficient contiguous gene deletion syndrome?
22q11.2 Di George syndrome - tbx1 HI causes heart defects and dysmorphology
give example of HI cancer gene?
TP53 - Thought to be typical two-hit model BUT LFS tumours analysed for LOH (loss-of-heterozygosity) show it occurs in ~60%, so ~40% of tumours have a presumably functioning second copy
what is a gain of function mutation?
what is a hypermorph and a neomorph?
- increase in gene expression or product develops new function
Hypermorph: an allele that produces an increase in quantity or activity of its product
Neomorph: an allele with a novel activity or product.
give examples of GOF mutations?
HD = CAG TNR exon 1 HTT polyglutamine tract 36 repeats or more. acquires novel deleterious function - deletions of region do not cause disease so GOF. forms inclusion bodies containing huntingtin which forms abnormal B sheets
DM1 - CTG toxic gain of function in DM1 and CCTG in DM2
Achondroplasia - inherited short stature FGFR3 tyrosine kinase receptor which negatively regulates bone growth. mutations activate the receptor, limiting bone growth. Gly380Arg) accounts for >99% of cases. high de novo mutation rate. FGFR3 mutations also cause thanatophoric dysplasia TD1 and 2, hypochondroplasia
BCR-ABL1 fusion gene in CML: t(9;22)(q34;q11) - novel tyrosine kinase. imatinib is TKI. MRD by RT-PCR quantifies levels of BCR-ABL1 mRNA transcripts in blood and bone marrow samples. determines treatment response.
CMT PMP22 - 1.5Mb duplication 17p11.2 - 80% of CMT1 cases. PMP22 codes for peripheral myelin protein. duplication generated by NAHR between sequences that flank the gene. reciprocal deletion causes HNPP
what is a dominant negative mutation?
mutations that reduce the function of the protein encoded by the normal copy of the gene
- only seen in heterozygotes and cause more severe effect than no gene product
- <50% residual function
-
give examples of dominant negative mutations
osteogenesis imperfecta
- COL1A1 or COL1A2 causes 90% of cases
- Haploinsufficiency (type 1): null variants and NMD results in milder phenotype as amount of collagen produced is reduced
- Dominant negative (Type 2): 80% caused by replacement of Glycine in Gly-X-Y in triple helix domain. disripts formation of triple helix and causes severe disease
Marfan - FBN1
- haploinsufficiency or dominant negative
- dominant negative: usually cysteine substitutions
Haploinsufficiency - nonsense/FS lead to NMD and decreased amount of fibrillin and aortic wall strength
what are the clinical symptoms of CF? (CF PANCREAS)
Chronic cough and wheezing
Failure to thrive
Pancreatic insufficiency (symptoms of malabsorption like steatorrhea)
Alkalosis and hypotonic dehydration
Neonatal intestinal obstruction (meconium ileus)/ Nasal polyps
Clubbing of fingers/ Chest radiograph with characteristic changes
Rectal prolapse
Electrolyte elevation in sweat, salty skin
Absence or congenital atresia of vas deferens
Sputum with Staph or Pseudomonas (mucoid)
what is the testing process for diagnostic CF case?
- screen for most common UK variants (Devyser, OLA, Elucigene) + MLPA (10% of CF variants are CNVs)
- If negative or only one variant identified do NGS for 27 exons
If rare homozygous variants in non-consanguineous individuals are detected what should be considered?
- deletion
- UPD7
Blood spots are collected on Guthrie cards at day 5 and analysed for the presence of immunoreactive trypsinogen (IRT) in duplicate – if levels of IRT are observed to be above the 99.5th centile, may be indicative of CF. What is IRT?
Immunoreactive trypsinogen - precursor to the enzyme trypsin. produced in pancreas and transported to small intestine where it is converted. In CF patients the mucous blocks the pancreatic ducts preventing trypsinogen from reaching the intestine and so Newborns with CF may have raised levels of trypsinogen in their blood for several months.
what is the CF testing pathway for a newborn with high IRT on first 5 day test?
what is the testing pathway for Babies with a FH, meconium ileus or echogenic bowel with a normal IRT?
screen 4 most common variants accounting for 80% of variants seen in the UK population. If a single variant detected or two variants (immediate referral to a CF specialist), screen for all variants.
2nd IRT on day 28 if first is raised. If no mutation identified but still high > CF suspected. If one mutation detected but still high > CF suspected. If one mutation detected and IRT normal on 2nd test = probable carrier.
Babies with a FH, meconium ileus or echogenic bowel with a normal IRT should be investigated according to clinical circumstances as well as being screened.
what 3 biochemical testing is available for CF?
- IRT
- sweat test - Individuals with CF will lose more Cl- in sweat than unaffected people. 90% of patients with have abnormal test
- Transepithelial nasal potential difference measurements assesses ion conductance in the upper respiratory epithelium: separately measures transport of sodium (Na+) and chloride ions (Cl-).
what is classical CF?
severe and chronic lung disease, cough, pancreatic insufficiency, pancreatitis, pulmonary infections, up to 20% of neonates meconium ileus at birth , high sweat chloride, congenital absence of the vas deferens (CBAVD). Caused by ‘severe’ mutations (no or reduced CFTR protein
what is mild CF?
a milder more variable disease resulting from mild mutations - some CFTR protein.
pancreatic sufficiency, onset usually after 10 years, no meconium ileus, lower sweat chloride levels and less severe respiratory disease
• Patients with a class 4 or 5 mutation have a later onset of respiratory symptoms as there is some function of CFTR (although less than wild-type CFTR).
which classes of mutations are pancreatic insufficiency associated with?
class 1, 2 and 3
levels of pancreatic function correlate well with CFTR genotype.
what is the Significance of the intron 8 poly(T) and poly TG tract?
- exon 9 is 90% skipped when the length of the tract is reduced to 5T
- The pathogenicity of the poly(T) tract is also mediated by the length of the adjacent poly(TG) tract, where long TG tracts are more likely associated with disease phenotype than shorter tracts (higher penetrance). Common TG lengths = 11, 12 or 13.
- Increased skipping of exon 9 is caused by TG12, TG13 and 5T
what is the significance of 5T in cis with p.(Arg117His) ? what about 5T in trans with a CF mutation or 5T/5T?
- modifies the expression
- 5T/Arg117His hets together with a typical severe pathogenic variant can cause classical CF of variable severity
- 7T/Arg117His hets (most 7T associated with TG11) and a typical severe mutation result in much more variable phenotype and can even be benign
- 9T/Arg117His hets are very rare and thought to be benign
- highly variable and more likely CFTR related disease eg. CBAVD, bronchiectasis/pancreatitis
which variant is 9T seen exclusively in cis with?
phe508del
when is reflex testing for 5T recommended?
- if arg117his identified
- Patients with bronchiectasis/pancreatitis with only one pathogenic mutation
- All males with infertility caused by obstructive azoospermia
what treatments are available for CF patients?
- daily airway clearance - physiotherapy
- mucolytic enzymes
- antibiotics - infections. do refex test for m.1555A>G predisposition to irreversible hearing loss and give other drugs if variant present
- Pancreatic enzyme replacement therapy
- lung transplant
- Gene Therapy:
Aminoglycosides - read-through PTCs by preventing ribosomal proofreading eg. gentamicin or Ataluren
Corrector therapy e.g. Lumacaftor. Facilitate proper maturation of CFTR and delivery to the membrane. Corrects for mutations that cause misfolding and degradation e.g. F508del
Potentiator therapy: e.g. Ivacafor: Improving CFTR channel function to correct for mutations that reduce gating efficiency e.g. G551D
- CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats) to remove mutated CFTR followed by homologous recombination with WT
-RNA editing: antisense oligonucleotides to replace deleted segments of mRNA
why might there be elevated neonatal IRT apart from CF?
neonatal stress (low Apgars), respiratory distress, hypoglycaemia, or serious congenital abnormalities such as trisomies 13 and 18
PPV is low for IRT
describe NBS for PKU?
- PAH gene - results in defects in phenylalanine hydroxylase enzyme
- metabolism of phenylalanine (Phe) to tyrosine (Tyr)
- buildup of phenylalanine causes ID
- mass spectrometry measures Phe and Tyr ratios
- sanger + MLPA in Bristol
- an advantage of genetic testing is some variants can be treated with BH4 instead of strict diet
- screen positive = special diet preventing severe disability
describe NBS for sickle cell disease?
- sCD means cells are unable to deform as they pass through narrow capillaries
- serious infection, chronic pain, pulmonary hypertension, proliferative retinopathy, organ damage, aplastic crisis, stroke or even death
- haemaglobin A accounts for 95% normal haemoglobin and accounts for two alpha and two beta chains. SCD is caused by mutation in B haemoglobin gene HBB.
- more common in people from tropical and/or sub-tropical regions where malaria was common as carrying a single sickle cell gene conferred an advantage
- screen positive = immunisations and antibiotics
- screened using HPLC, isoelectric focussing and capillary electrophoresis
- caused by HBB gene, c.20A>T p.Glu6Val detected by ARMS or pyrosequencing
- HBB, HBA1 and HBA2 genes can be tested for thalessaemia
describe MCADD NBS?
- problems breaking down fats leading to illness or death
- AR ACADM
- Single common mutation c.985A>G (p.Lys304Glu) is responsible for 88%
- screen positive infants are put on special diet
- mass spec of C8 levels relative to c10
- targeted testing of the c.985A>G followed by extended screening
what is the MOI and mutation for FRAX?
what FMR1-related disorders are there?
X linked dominant, variable penetrance, 5’ UTR CGG repeat >200 causes methylation gene silencing
- small proportion of cases due to point mutations or partial or whole gene deletions or duplications
fragile X tremor ataxia syndrome/ primary ovarian syndrome, fragile X syndrome
CAG repeats caused by polyQ glutamine, give three examples, other than spinocerebral ataxias. do these mutations occur in coding or non coding regions?
- HD - HTT gene
- SBMA - XLR - AR gene (also causes androgen insensitivity syndrome)
- dentatorubral-pallidoluysian atrophy (DRPLA) - CAG expansion in ATN1 gene
All exonic! GOF - CAG repeat expansions result in polyglutamine aggregates
what are the repeat sizes for FRAX?
N up to 45
46-58 int
premutation 59-200
mutation >200
what is the phenotype of Fragile X full mutation in males? and females? why is there a variable phenotype in females?
- moderate to severe ID and social impairment, large ears, macroorchidism, hypermobile
- apparently normal (about 50%) to mild to moderate mental and social impairment
- skewed x inactivation
what is the phenotype associated with FRAX premutation in females? and in males?
- POI - cessation of menses before 40 and early menopause
- occurs in 20% of cases
MALES
- late-onset progressive cerebellar ataxia and intention tremor
- onset usually >60 years, penetrance ~50%
what is FRAXE? what is the mutation and phenotype?
- distal fragile site Xq28
- GCC 5’ UTR expansion of FMR2
- phenotype is considerably less severe than the Fragile X of FRAXA and lacks the specific syndromic features
- no phenotype linked to premutation
what are limitation of PCR for FRAX?
- will not detect mosaicism or rare point mutations
what is an intermediate FRAX allele?
46-58 repeats
often transmitted stably, but may show increasing unstable transmission with larger sizes.
stability correlates with correlate with the presence of two or more interspersed AGG motifs within the CGG tract
what are FRAX premutations?
59-200 repeats
unmethylated
- pcr can detect smaller alleles
- expansion to full mutation exclusive to females and usually if >90 repeats
what % of FRAX full mutation patients are mosaic?
20% of full mutation patients also show some mosaicism for a premutation
some are full mutation methylation mosaics
describe Amplidex for FRAX testing?
TP-PCR method
allows determination of CGG repeat length up to 200 CGGs and non-quantitative detection of alleles greater than 200 CGGs (i.e. full mutations)
- can detect interrupting AGG sequences and detect size mosaics
- A methylation sensitive kit is also available for the detection of methylation status in the FMR1 gene
- able to detect full mutations and resolve female zygosity (one peak on PCR - homozygous or missed full mutation?)
what testing methods are available for FRAX? what are advantages and disadvantages
- direct PCR - quick and cheap. only detects up to 120 repeats, preferential amplification, doesn’t detect premutation/full mutation mosaics. males with no peaks and females with one peak should have further testing.
- southern blot - detects all sizes and methylation but laborious, large amounts of DNA required, doesnt have the resolution to detect size
- Amplidex - detects normal, premutation and full mutations +some detect methylation, AGG repeats but expensive as a first-line test
- linkage - STR markers. may be useful in prenatal eg. if southern blot fails.
- NGS - SNVs, deletions, duplications
what is genomic imrprinting?
epigenetic phenomenon that causes genes to be expressed or not, depending on whether they are inherited from the mother or the father.
This leads to differential expression of the paternal and maternal alleles in specific tissues or developmental stages.
what is an imprinting control region?
differentially methylated region that regulates expression of genes in an imprinted domain
ICRs show parent-of-origin-specific epigenetic modifications, including histone modification or DNA methylation within differentially methylated regions (DMRs), which determine whether or not the genes within the imprinted domain are expressed
The imprinting marks at ICRs must be reset during gametogenesis to reflect the sex of the parent for the next generation
name the 5 molecular changes that result in imprinting disorders?
- UPD - if chromosome is imprinted, UPD changes gene expression eg. two active alleles or two inactive. recurrence risk <1%. prenatal testing should be offered to balanced translocation carriers of imprinted chromosomes.
- chromosomal rearrangements - deletions, dups, translocations - deletion of ICR leads to loss of regulation of imprinted region, duplication may lead to overexpression of gene
- mutation - active gene mutation leads to parent-of-origin phenotype
- epimutation - eg. methylation hyper or hypo alters expression at differentially methylated regions
Disruption of paternally-expressed genes causes what phenotype? and maternal?
what features are common to most imprinting disorders?
undergrowth
overgrowth
growth, metabolism and development
which 6 imprinting regions are associated with disease?
6q24- transient neonatal diabetes
7 - russel-silver syndrome,
11p15.5 BWS/RS
14q32 mat UPD14 = temple syndrome, pat14 = wang syndrome
15q11.2, PWAS
20q13.2: UPD chromosome 20
how might assisted reproductive technologies impact imprinting disorders?
ART may prevent the proper erasure, establishment, and maintenance of DNA methylation
what causes transient neonatal diabetes? what are the clinical synptoms?
= intrauterine growth retardation (IUGR) and hyperglycaemia due to insulin deficiency. normally maternal DMR is silenced and pat is expressed.
caused by overexpression leading to twice normal dosage eg. patUPD6, paternal 6q24 duplications or hypomethylation of mat region.
what are the clinical features of PWS?
overgrowth (pat deletion)
hyperphagia
behavioural difficulties
mild ID
hypotonia - floppy baby
hypogonadism
what are the clinical features of AS?
- smiley
- severe ID and speech
- undergrowth
- gait ataxia
- epilepsy/seizures
where is UBE3A expressed and what does it code for?
expressed from mat allele in brain - crucial for normal development of synapses
what is the recurrence risk for a mat IC deletion in AS? what affects this recurrence risk?
what about pat IC deletion in PWS?
50% if inherited from mum
50% if inherited from dad
list causes, % of cases and recurrence risks of PWS mutations?
de novo paternal deletion = 75% of cases and <1% recurrence
mat UPD = 30% of cases <1% recurrence
IC deletion= 15 % cases and 50% recurrence risk
Imprinting defects 1% cases 1% recurrence
list causes, % of cases and recurrence risks of AS mutations?
de novo mat deletion = 75% of cases and <1% risk
UBE3A mutation = 10% cases and 50% risk
IC deletion = 10% cases and 50% recurrence
pat UPD 2% of cases <1% recurrence
imprinting defect = 3% cases and <1% recurrence
no identifiable cause in 15% of cases
how do PWAS deletions occur
NAHR between HERC2 copy repeats
• The 15q11-13 region contains 5 common break points which comprise breakpoints 1 through 5 (BP1-BP5)
Deletions involving either BP1 or BP2 and BP3 are most common
describe the testing pathway for PWAS?
- MS-MLPA or MS-PCR for methylation analysis. If normal, unlikely to be PWS but may be AS due to mutation so do sequencing. hypomethylation = AS hypermethylation = PWS. MS-MLPA detects deletions and IC deletions. not able to distinguish UPD from imprinting defect. then:
- Deletion analysis - MS-MLPA, Microsatellites or FISH if IC deletion check parents as 50% recurrence. if het deletion do chromosomes in proband and parents. If no deletion do microsatellites to check UPD. If normal could be imprinting defect with no molecular change.
In cases of UPD15, parents should be investigated to rule out robertsonian translocation.
IF no deletion or UPD identified in patient with abnormal methylation > send to specialised lab for IC deletion testing as significant recurrence risk or may be imprinting defect (low risk)
what cytogenetic methods with detect 15q11q13 dels?
FISH karyotype array
methylation testing also required to confirm diagnosis. MS-PCR or southern blot confirms diagnosis but not the disease mechanism.
what types of UPD is microsatellite analysis able to detect for PWAS?
hetero and isodisomy
does absence of methylation rule out PWAS? what about AS?
PWAS - almost 99% have abnormal methylation
AS - no 20% have normal methylation eg. UBE3A mutation, mosaics, 10% have unidentified cause - may be undetected UBE3A mutation or another causes
what differential diagnoses are there for PWAS?
PWS:
- SMA, myotonic dystrophy
- mat UPD 14 Temple syndrome - hypotonia and obesity
- hyperphagic short stature
AS:
rett syndrome
Mitochondrial encephalopathy
what current treatments exist for PWAS?
- behavioural/physical therapy
- drugs for seizures
- specialised equipment for feeding difficulties
- growth hormone therapy
name how a future treatment may restore UBE3A function in AS?
activate the paternally derived UBE3A gene by inhibiting paternal antisense strand by:
- CRISPR/Cas9 system can be used to target the SNRPN gene to inhibit the expression of UBE3A-ATS and thus enable the UBE3A gene to be expressed
- In mice - truncate UBE3A-ATS by inserting a poly(A) cassette to allow pat gene to be expressed
ADD TO CARDS which genes are implicated in telomeric 11p15 imprinting cluster 1, which allele (mat or pat) are they expressed from and what is their function?
H19 (mat expressed) noncoding RNA
IGF2 (pat expressed) growth promoter
CTCF binds maternal unmethylated ICR1 to block IGF2 promoter and enhancer interactions allowing H19 to be maternally expressed.
conversely, methylated pat ICR1 prevents CTCF binding- IGF2 expressed and H19 silenced
ADD TO CARDS which genes are implicated in centromeric 11p15 imprinting cluster 2, which allele (mat or pat) are they expressed from and what is their function?
KCNQ1 (mat expressed) - voltage gated potassium channel
KCNQ1OT1 (pat expressed)- a non-coding RNA with antisense transcription to KCNQ1
ICR2 is maternally methylated and includes KCNQ1OT1 promoter
CDKN1C (mat expressed) – encodes a cyclin dependent kinase inhibitor
what are the clinical features of BWS? can it be diagnosed prenatally?
- overgrowth, high insulin - low blood sugar, macroglossia, tumours eg. Wilms tumour
- USS features eg. Polyhydramnios but needs molecular diagnosis to be diagnosed
ADD TO CARDS what are the main genetic causes, frequency and recurrence risks of BWS?
- pat chr11 UPD - 25% of cases, <1% recurrence. mosaicism possible
- ICR2 mat hypomethylation - 60% of cases, <1% recurrence - mosaicism possible
- IC1 mat hypermethylation, 10% of cases, <1% - mosaicism possible
- duplications, deletions, translocations, 2% cases, up to 50% recurrence depending on parental transmission
- CDKN1C mat mutations 5% cases (50% de novo, 50% inherited) 0% or 50%, depending on the sex of the parent contributing the affected allele
what is the main testing method for BWS? what other methods are there?
- MS-MLPA
- CDKN1C sanger
- array CGH, SNP array, karyotype, FISH, MLPA
what are the clinical features of russel-silver syndrome? can it be diagnosed prenatally?
- prenatal and postnatal growth retardation
- macrocephaly
- protruding forehead
- body assymetry
- feeding difficulties
- low muscle mass, BMI
- developmental and speech delay
what are the main genetic causes, frequency and recurrence risks of russel silver?
- pat IC1 hypomethylation, 60% cases, low recurrence
- deletions/duplications -1% cases, up to 50 % recurrence depending on parent eg. maternal duplication
- maternal CDKN1C GOF point mutation - 50% recurrence
- paternal IGF2 LOF point mutation - 50% recurrence
- mat UPD 7, 10% cases, <1% recurrence
what are the main testing methods for russel-silver syndrome?
- MS-MLPA, array, WES, MLPA
give examples of genes that cause several diseases (clinical heterogeneity)
- PMP22 17p11.2= peripheral myelin protein 22
1.5 Mb deletion causes HNPP Hereditary Neuropathy with Liability to Pressure Palsies. muscle weakness and atrophy.
CMT1A = PMP22 duplication 1.5Mb duplication/GOF mutation. progressive muscle weakness. - FGFR3 - tyrosine kinase receptor that negatively regulates bone growth. GOF mutations cause hypochondroplasia, achondroplasia, TD, Muenke Syndrome. somatic activating mutations cause bladder cancer.
- AR gene. polyglutamine expansion causes SBMA. muscle weakness. onset decreases as expands. het females usually unaffected.
androgen-insensitivity syndrome- complete, partial and mild
give examples of diseases caused by several genes? (genetic heterogeneity)
cardiomyopathy - shortness of breath, chest pain progressive heart failure to sudden cardiac death
Most common genes mutated= MYH7 (40%), MYBPC3 (40%), TNNT2 (5%), TNNI3(5%), TPM1 (5%)
Incomplete penetrance and variable expressivity
Caused by dominant negative, haploinsufficiency and GOF mutations
what is Pleiotropy
occurs when one gene influences two or more seemingly unrelated phenotypic traits
what is a polygenic risk score?
quantify cumulative effects of no. of genes, which may individually only have small effect on susceptibility; score reflects sum of all known risk alleles, weighted by how risky each variant known to be
predict persons likelihood of displaying a trait
• Genome-wide data allows access to millions of common genetic variations associated with these conditions; risk score for each person calculated with aim to inform clinical management
what are the benefits of polygenic risk scores?
• Helpful in targeting people at higher-risk of conditions where increased surveillance + preventative treatment/surgery available
• Help to match drugs in clinical trials to individuals who are most likely to benefit from them
- personalize preventative measures
what are the drawbacks of polygenic risk scores?
• Not diagnostic: high risk score doesn’t mean person will definitely develop condition + vice versa
• Results need to be communicated carefully to minimise risk of confusion
• Requires consent for genetic testing; may be difficult to obtain
• Accuracy; majority calculated from European DNA sequences; less accurate for other populations
why are repeat expansions known as dynamic? what causes changes in TNR numbers?
premutations: can expand or contract when transmitted
Dynamic mutations also show somatic instability, which accounts for some of the phenotypic variability.
strand slippage during DNA replication in actively dividing cells
what mutation causes Motor neurone disease (MND) and frontotemporal dementia (FTD) ?
AD c9orf72 hexanucleotide repeat GGGGCC expansion
what mutation causes DM2? what are symptoms? what is the repeat size range?
tetranucleotide repeat CCTG in intron 1 ZNF9
muscle weakness, muscle pain,
Full penetrance, mutation alleles >75 repeats, up to 11,000 repeats
give examples of triplet repeat disorders caused by LOF mutation?
Fragile X Syndrome (both FRAXA and FRAXE) and Friedreich Ataxia (FRDA)
expansions result in loss of gene product or function
Point mutations are rare in affected patients (<1% Fragile X cases, ~2% FRDA cases)
describe freidrich ataxia, what the disease mechanism and mutation is, clinical features
- AR so anticipation not observed, carriers unaffected
- most common form of ataxia with 1/50 carrier frequency
- progressive ataxia (co-ordination, balance and speech) muscle weakness and HCM
- homozygous expansion of a polyE glutamic acid GAA repeat in intron 1 of frataxin (FXN)
- 2% of patients have expansion and LOF mutation- Nonsense, missense, frameshift, and splicing defect in trans
- mutation causes defective transcription of the FXN gene, leading to deficiency of frataxin (mitochondrial protein)
-PCR, TP-PCR and southern blot can be used to test & MLPA + sequencing if strong clinical suspicion and only one expansion identified
give examples of Coding GOF polyglutamine disorders (CAG repeat expansions) ?
SBMA, HD, DRLPA, SCAs
give examples of non coding GOF disorders?
DM1, DM2, SCA 8 + SCA 12
what mutation causes DM1? what are clinical features?
what are the repeat sizes?
-GoF - Haploinsufficiency caused by CTG expansion
-RNA GoF- CUG expansions in 3’ UTR fold into RNA hairpins that accumulate in ribonuclear foci
-encoding DMPK protein – serine-threonine kinase, mainly expressed in heart + skeletal muscle
muscle weakness + wasting ,cataracts, cardiac defects, floppy baby + respiratory distress
normal = up to 36 repeats (same as HD)
premutation 37-50
full penetrance alleles 51-150
juvenile > 150
congenital = >2000
what is the mutation that causes HD? what are the repeat ranges? what are clinical symptoms? what testing can be offered to parents who do not want to know disease status?
CAG expansion in exon 1 HTT, higher repeats = earlier onset
<27 = normal
27-35 = intermediate
36-39 = reduced penetrance (some elderly unaffecteds)
>39 = expansion
>60 = juvenile
- chorea, dystonia (muscle spasm), psychiatric disturbance, involuntary movements, cognitive decline
- large expansions exclusively paternal
- exclusion testing can be offered where parent doesn’t want to know status (25% risk to child)
what therapies are in development for TNR disorders?
- Antisense oligonucleotides- IONIS-HTT: nhibit expanded mRNA to reduce concentration of mutant HTT protein
- RNAi- mouse model of SCA1, with adeno-associated virus (AAV) vectors expressing short hairpin RNAs that efficiently reduced expression levels of mutant protein
- Protein aggregation inhibitors e.g. (2)-Epigallocatechin-3-Gallate (EGCG) for HD
describe clinical features of FSHD, the mutation responsible and how to test?
FSHD - Facioscapulohumeral muscular dystrophy
- 95% of cases are FSHD1
- muscle weakness
- FSHD1 caused by AD contraction of D4Z4 repeats (contains DUX4 gene)
<10 repeats = pathogenic
- results in reduced transcription of DUX4
- diagnosed by southern blot due to large size using restriction enzymes
- FSHD2 caused by SMCHD1 mutations and diagnosed by bisulphite PCR and NGS
give examples of poly-alanine expansion disorders?
XLMR - x-linked mental retardation ARX gene
Hand–foot–genital syndrome - HOXA13
List three differences between the nuclear and mitochondrial genomes
mitochondrial genome is circular and double-stranded
mitochondrial genome contains 37 genes
There are no introns - only 3% is non-coding
mitochondrial mode of inheritance is strictly maternal
Each cell can contain 100 to 10,000 copies of the mt genome but only one copy of nuclear
define homoplasmy, heteroplasmy and mt genetic bottleneck
what is the threshold effect?
mtDNA can all be identical (homoplasmy)
mixture of two or more mt genotypes (heteroplasmy)
The percentage level of mutant mtDNA may vary among individuals within the same family, and also among organs and tissues within the same individual (mt genetic bottleneck).
• The threshold effect is that there is a level of variant mtDNA that can be tolerated by the cell, however above a certain level (which is tissue specific) oxidative phosphorylation cannot be maintained and disease manifests
Describe 3 considerations for interpretation of pathogenicity unique to mtDNA variants
Homoplasmic variants inherited from a homoplasmic clinically unaffected mother are unlikely to be pathogenic.
mitomap - phenotypes and genbank frequency
The level of heteroplasmy must be accurately determined and interpreted in the context of the tissue
Evolutionary conservation and functionality eg. MitoTIP
why is mitochondrial genetic testing particularly difficult?
- variant differs by tissue type and age of patient
- Some pathogenic variants (e.g. m.3243A>G) are lost from tissues such as blood that undergo rapid mitotic division
- a neg result doesnt mean the patient doesnt have variant
- DNA extracted from the affected tissue is preferred
- Genotype-phenotype correlations are poor
Clinicians have referred an adult presenting with optic neuropathy to the highly specialised mitochondrial diagnostic service. Describe the appropriate testing pathway and any relevant candidate genes and variants for targeted analysis
MT-ND1 m.3460G>A , MT-ND4 m.11778G>A and MT-ND6 m.14484T>C
targeted testing of 3 common mutations eg. pyrosequencing
mitochondrial WGS
DNAJC30 single gene sequencing
referred from Consultant Ophthalmologist, Neurologist or Clinical Geneticist
clinical assessment, family history, biochemical testing, histopathological examination (muscle biopsy) and direct molecular testing
Name the gene responsible for encoding mitochondrial DNA polymerase
POLG
what is MELAS? what is the mutation? what sample types should be used?
Mitochondrial encephalopathy, lactic acidosis and stroke-like episodes
MT-TL1 m.3243A>G
urine or muscle best samples
ADD TO CARDS what is leigh syndrome? what is the mutation?
MT-ATP6 m.8993T>C (also MT-ND1, 4 and 6)
Encephalopathy, lactic acidosis
what techniques are used for mt-DNA testing? what is the best sample for testing?
common mutations = sequencing/pyrosequencing
rearrangements such as deletions = long range PCR. multiple mtDNA deletions may be due to nuclear gene mutation such as POLG for example
RT-PCR - copy number and WGS if increased copy number as likely to be a mtDNA mutation
if not mt DNA gene need to consider nuclear sequencing - encodes mt subunits and mt genome maintenance
muscle is the best tissue to test
why is mtDNA testing particularly difficult for prenatal diagnosis? what methods can be undertaken to prevent mt disease transmission?
- heteroplasmy difficult to interpret
- % heteroplasmy in CVS may not represent other tissues and levels may change during development
- difficult to predict severity of disease
- ooctye donation or nuclear transfer where the nuclear genome from the oocyte or embryo of an affected woman is transplanted into a donor enucleated oocyte or embryo with healthy mt. nuclear DNA can be transferred between unfertilised oocytes using polar body transfer or maternal spindle transfer or between fertilised zygotes using pronuclear transfer
what is a chromosome instability/breakage syndrome?
inability to repair a particular type of DNA damage
usually AR
hypersensitive to certain drugs leading to chromosomal rearrangements
increased predisposition to cancer
diagnosed through cytogenetics biochemical and molecular methods
Fanconi Anaemia (FA) - what are the genes responsible, clinical features and testing?
most common breakage syndrome
FANCA is the most common cause, but 15 different groups
mostly AR, but also XL (FANCB - males affected) and AD
growth retardation, dev delay, skeletal malformations, increased susceptibility to leukaemia, anaemia
Mutated cells have deficient ability to excise UV-induced pyrimidine dimers from the cellular DNA - it leads to double-strand breaks in the S phase of the cell cycle
Testing: culture cells with cisplatin (interstrand cross-linking agent) causes breakage and sequence FA genes
Bloom syndrome-what is the gene responsible, clinical features and testing?
AR BLM gene - DNA helicase tumour suppressor gene that suppresses inappropriate recombination
sun sensitive rash, growth deficiency, immunodeficiency, predisposition to cancer, infertility in males
increased SCE level
gene sequencing and count SCE frequency in 20 metaphases
Ataxia Telangiectasia (AT) - what is the gene responsible, clinical features and testing?
chromosomal breakage
AR disorder
ataxia-jerky movements, immunodeficiency, cancer predisposition - leukaemias
ATM gene sequencing - usually compound het mutations but also some dominant negative heterozygous missense variants. Gene codes for a kinase which signals ds breaks and nonhomologous exchange.
cytogenetics - radiation causes chromosome breakage
Xeroderma Pigmentosum - what is the gene responsible, clinical features and testing?
sun sensitivity, cancer predisposition, hearing loss, cognitive impairment,
genetic sequencing- 8 genes involved in nucleotide excision repeair (NER) eg. XPC, ERCC2 and POLH
caused by UV radiation damaging genes that control growth and division - cells die or grow uncontrollably leading to cancers
why might there be an abnormal phenotype in a patient with a balanced rearrangement? (8 answers)
- gene disruption (most common cause) - truncated or no protein produced. may unmask recessive condition if SNV in other allele. FISH can detect this and NGS with paired end reads. can help to refine breakpoints. can create fusion genes.
- cyrptic imbalance - more complex (>3 breaks the more likely to have cryptic imbalance). may have dels, ins or inv near breakpoint or not at breakpoint site. may be inherited or de novo. phenotype depends on size of cryptic imbalance.
3) position effect - moves gene away from cis elements such as enhancer, inhibitor or closer to enhancer of another gene causing cancer, regulatory element may be moved closer to another gene altering expression levels and euchromatic material may be moved to heterochromatic region causing silencing and vice versa.
4) imprinting disturbance - imprinted region moved away from IC
5) UPD - robertsonian and translocation carriers may have offspring with UPD. relevant if genes are involved in imprinting or unmasks recessive allele eg. isodisomy. very rare
6) X;autosome rearrangements - males always infertile. due to spermatogenic arrest. Females - if normal X inactivated it is balanced. If abnormal X with XIC inactivated it will result in some autosome being inactivated too and translocated X will not be inactivated causing abnormal dosage. also if derivative autosome contains XIC it will also be unbalanced. t(Y;autosome) carriers usually infertile due to spermatogenic arrest.
7) mosaicism - may be mosaic for unbalanced karyotype in certain tissues Eg Pallister-Killian - normal in blood. or may have interchange trisomy followed byl loss of normal chromosome to give balanced karyotype in blood
8) may be IF and cause is due to something else
why does a balanced translocation result in infertility?
disrupt gamete formation especially in males
failure of pairing of homologous regions in the quadrivalent formed at meiosis I by a reciprocal translocation, which then interfere with the X-Y bivalent disrupting meiosis.
robertsonian translocation - p arms interfere with X;Y bivalent
fertility may vary between males in same family
what proportion of moderate to severe ID is genetics, environment and unknown? what % of population have ID? why is a diagnosis useful?
up to 40% genetic
up to 30% environmental
rest unknown
1-3%
prognosis, clinical care and educational needs, support groups and prenatal diagnosis.
currently incurable
how does array CGH compare to karyotype for ab USS findings?
Increases detection of chromosome abnormalities by up to 6% compared to karyotype for USS findings
what is the diagnostic yield of exome sequencing for patients with severe ID
25% - usually done after a normal array or WGS
how does use of array as first line test for DD, LD, autistic spectrum disorders or multiple congenital anomalies improve detection rate compared to karyotype?
use of arrays increased the detection rate from approx 3% with karyotyping to approx 15-20% and represent a 100 fold higher resolution approach
what is a marker chromosome?
AKA: Small supernumerary marker chromosome (sSMC)/ Extra structurally abnormal chromosome (ESACs)/Accessory chromosomes
structurally abnormal chromosome that cannot be identified by conventional banding alone and is equal to or smaller in size than chromosome 20 of the same metaphase spread
majority derived from acrocentric chromosomes
Can be inverted duplication, ring, centric (have centromere), neocentric (new centromere)
some produce abnormal phenotype but more than half don’t- clinical outcome difficult to predict - especially prenatally.
If identifyable use der(chr) instead of marker
50% are mosaic - almost all pallister-killian are mosaic
may cause infertility by interfering with meiosis
80% are de novo - usually abnormal phenotype & low recurrence risk
<0.1% of live births
23% inherited but most without clinical signs (mostly maternal)
• Very small markers are prone to loss during cell division – mosaicism often seen
which chromosome accounts for majority (30%) of marker chromosomes?
what other syndrome accounts for large proportion of SMCs?
15
- 50% have abnormal phenotype when PWAS region is present
- emanuel syndrome der(22)
how might marker chromosomes arise?
- numerical monosomy or trisomy rescue, gamete complementation or unbalanced structural rearrangement. Can be meiotic or mitotic.
- inverted duplication chromosomes -U-shaped exchange - crossover mistakes of chromatids of two homologous chromosomes during meiosis
(monosomy rescue likely UPD) - test if derived from imprinted chromosome eg. 6,7,11,14,15&20
what factors influence if a phenotype results from a marker chromosome?
- size and origin or euchromatin
- mosaicism percentage
- UPD
Generally, smaller markers with low or no euchromatin → low risk, larger markers with euchromatin → high risk. But interpret with caution, as presence of euchromatin does not necessarily lead to clinical effects.
If the marker is inherited from a normal parent, this is likely to indicate a benign marker, BUT need to consider levels of mosaicism and affected tissue types. A number of familial cases have been reported where a parent carries a marker in mosaic form with no phenotypic effect, while their child carries the same marker in non-mosaic form and is severely affected.
give examples of known clinical syndromes associated with marker syndromes? what are clinical features?
1) pallister killian tetrasomy 12p. (12% of markers)- severe MR, seizures, diaphragmatic hernia + facial dysmorphism. blood normal, detectable in fibroblast tissue. false negative rate in CVS/prenatal is 10% due to mosaicism
2) emanuel syndrome + der(22)t(11;22)(q23;q11) . Trisomy for
11q23-qter and
22q11-pter
severe MR, cleft palate, Micrognathia (undersized jaw), renal/heart defects
3:1 segregation of recurrent t(11;22) resulting in only viable tertiary trisomy
Carriers of the balanced translocation have 10% chance of conceiving a child with the syndrome
give examples of markers involving sex chromosomes?
variant turner - 46, X + SMC or may be in addition to XX/XY phenotype depends on if XIST - if absent, clinical complications may arise
idic Y/inv dup Y
46, X+SMC - absence of SRY = turner syndrome phenotype with increased risk of gonadoblastoma, if SRY present but Yq- = male infertility
how do you investigate marker chromosomes?
- FISH, aCGH + karyotype
- acgh - useful to classify as normal for first-line test. FISH may be required if additional material present to show location.
- FISH able to show if centromere is present
- aCGH may not detect low-level mosaicism
- SNP array will detect UPD
- can use agNOR staining to see if acrocentric
what is Chromothripsis?
up to thousands of clustered chromosomal rearrangements occur in a single event in localised and confined genomic regions in one or a few chromosomes
catastrophic single event producing complex chromosomal rearrangement
causes rare disease, infertility and cancer
chromothripsis derives from chromosome shattering followed by the random restitching of chromosomal fragments with low copy-number change
what is chromoanagenesis?
erroneous DNA replication of a chromosome through serial fork stalling and template switching with variable copy-number gains
what is chromoplexy?
an accumulation of translocations involving several chromosomes
no copy-number alterations
what are the 5 classes of CFTR mutations?
- no function protein produced
- misfolding eg. F508del - lumacaftor
- channel gating eg. G551D - Ivacaftor
- faulty channel conductance
- normal protein created but insufficient quantity
