Session 3 - Postnatal testing Flashcards
Define Anticipation.
Give examples of 3 diseases that show anticipation
The phenomenon in which the severity of disease increases as it is passed down the generations.
FraX, DM, HD, DRPLA, SCA1, SCA3, SCA7.
List possible mechanisms of repeat expansion in diseases showing anticipation.
Replication slippage.
Incorrect reassembly of Okazaki fragments
Formation of hairpin loops causing the replication fork to stall.
Unequal crossing over.
Is anticipation observed in polyglutamine or polyalanine disorders?
Polyglutamine.
What three biases can give a false impression of anticipation?
Preferential ascertainment of parents with late onset of disease.
Preferential ascertainment of children with severe early onset disease.
Preferential ascertainment of parent-child onset at the same time.
Define Age-related mosaicism
The accumulation of somatic mutations over time.
Give examples of age-related mosaicism.
X-chromosome aneuploidy/loss. Caused by premature centromere division during mitosis.
Cancer
Define Variable expressivity
Phenotype is expressed to different degrees among individuals with the genotype
Give examples of variable expressivity
Marfan syndrome - FBN1.
NF1
Waardenburg syndrome
Define penetrance.
The likelihood of experiencing a disease if you have a pathogenic mutation.
Give examples of penetrance
TOR1A - early onset dystonia - common mutation c,907_909delGAG present in >99% of individuals - only 30% express disease.
Define sex-limiting
Phenotype is limited to a single gender - only expressed in males or females.
Give an example of a sex-limited disease.
Familial precocious puberty in males. LHCGR mutations
Define epistasis
The presence of a variant in one gene determines the expression of a variant in a non-allelic gene
Give an example of epistasis.
Bombay phenotype ‘overrides’ the usual A/B dominance/co-dominance - if an individual is homozygous for the Bombay variant their blood group will be O.
Albinism overrides hair colour genes.
Define pleiotropy
One gene causes mutliple, seemingly unrelated phenotypes.
Give an example of pleiotropy
PKU - caused by mutations in PAH, require to turn phenylalanine into tyrosine. Lack of tyrosine = lack of downstream compound, melanin, hence the albinism.
Define the following mutation types:
- Amorphic
- Hypomorphic
- Hypermorphic
- Antimorph
- Neomorph.
- No protein product.
- Partial loss of protein expression
- Partial gain in protein activity - GoF
- Mutation acting as antagonist to usual function - dominant negative.
- Mutation gives protein a new function.
Give examples of X-linked dominant diseases
Non-lethal in males:
Alports (COL4A5 - deafness, RP, renal problems)
Hypophosphataemia (PHEX - loss of phosphate through kidneys = vitamin D3 deficiency and rickets)
FraX
Lethal in males:
IP - NEMO - can be in in 47,XXY
Rett syndrome - MECP2 (CDKL5, FOXG1) - can be seen in 47, XXY and in MECP2 duplication.
XLD - males unaffected
Lack of protein is not disease-causing - mutant protein is.
Or, only mutant protein is fine, WT + mutant causes problems. Craniofacial synostosis syndrome (EFNB1)
Give examples of XLR diseases.
Dystrophinopathies - DMD/BMD Androgen Insensitivity Haemophilia A/B SBMA Fabry XL-RP Huner syndrome Colour-blindness.
List clinical features of DMD
Calf hypertrophy Progressive symmetrical muscle weakness Joint contractures Gower sign Age of onset 2-5years Wheelchair bound by early teens. Death by 3rd decade
List clinical features of BMD
Later onset muscle weakness
Death in mid-40s
Often remain ambulatory til 20s
What serum metabolite can be used to diagnose DMD/BMD/DCM? What results would you expect to see for each?
Increased CK in the blood.
DMD 10x normal
BMD 5x normal
DCM ~normal
What is the advantage of performing genetic testing for DMD/BMD/DCM?
Don’t need to take a muscle biopsy.
Summarise the testing strategy for dystrophinopathies.
del/dup analysis
no variant then sequencing
no variant then western blot of tissue sample and IHC
What is special about the DMD gene?
It has three promoters - B (brain), M (muscle) and P (purkinje). These promoters produce a different transcript in different tissues. Each transcript has a unique exon 1, and the same 78 subsequent exons.
What is DMD-associated cardiomyopathy caused by?
Mutations in exon 1 associated with the M promoter. Skeletal muscle effects of the disease are not observed, as transcription of B and P transcripts in other tissues is increased to compensate.
Describe the mutation spectrum seen in DMD/BMD
Most are del/dups of >1 exon: DMD 60-65%, BMD 80-85%
Two deletion hotspots exist:
- central region, exons 44-53 - 80% of DMD del, 20% DMD dup
- 5’ region, exons 2-20 - 20% of DMD del, 80% DMD dup.
What can be used to predict pathogenicity in DMD/BMD?
Frameshift hypothesis - out of frame deletions are most likely to cause DMD (more severe phenotype) than in-frame deletions.
What factors complicate molecular diagnostics in DMD/BMD?
High new mutation rate (1/3 cases)
Germline mosaicism in mothers of patients. Recurrence risk is 5%
High recombination rate - 10%.
What are possible treatments for DMD/BMD?
STOP codon read-through - Gentamicin treatment
Exon skipping using antisense oligos to promote skipping of exon(s) and restoration of reading frame.
What causes Kennedy disease (SBMA)?
Repeat expansion in the AR gene (>38 repeats of the polyglut tract causes fully penetrant disease)
What are Haemophilia A and B caused by?
Mutations in F8 and F9, respectively.
What is the most common mutation, seen in 45% of haemophilia A patients?
inversion of intron 22.
What are the diagnostic sensitivities of testing methods used to diagnose Haem A and Haem B?
Haem A - 98%
Haem B - 90%
Define haploinsufficiency.
The loss of half of the amount of gene product is sufficient to cause disease.
What type of genes show haploinsufficiency?
Highly expressed genes - lots of gene product needed, so one copy isn’t enough.
Dosage sensitive genes - proteins that are part of a quantitative signalling system etc.
Imprinted genes
List examples of haploinsufficient genes in single gene disorders
Hypertrophic cardiomyopathy: MYH7, MYBPC3, TNNT2, TNNI3 - 4 gene assay will detect 50% of all cases of HCM
Alagille syndrome - JAG1 - multisystem phenotype
Aniridia (PAX6)
Autosomal dominant optic atrophy (OPA1)
HNPP/PMP22 - duplications cause CMT1A, mutations cause HNPP.
List examples of haploinsufficient genes in contiguous gene deletion syndromes
DiGeorge - TBX1
Williams - ELN
WAGR - PAX6
List examples of haploinsufficient genes in cancer
TP53
BRCA1
PTEN
It is now suspected that a single mutation in these genes may be sufficient to cause cancer. Two-hits not required.
What is a gain of function mutation?
A Hypermorph or Neomorph; result in an increase in gene expression or increase in activity of the gene product
Give some examples of Gain of Function mutations
PTPN11 - Noonan syndrome - activation of RAS/MAPK pathway
HTT - Huntington disease - protein aggregation
PMP22 - HMSN/CMT - Overexpression
BCR-ABL1 - CML - Chimeric protein
Where in HTT is the unstable CAG repeat?
Exon 1
What is a normal sized allele in HD?
Normal <36
What evidence is there that HD is a GoF disease?
Patients with chromosomal deletions do not manifest
Homozygotes are as affected as heterozygotes
Phenotypically normal individuals have been identified with a translocation with a breakpoint in HTT
HD levels of polypeptide from the normal and mutant alleles are the same.
Describe the molecular pathogenesis of HD
Mutant HTT forms abnormal protein structuers and is truncated by Caspase-6 cleavage to produce toxic N-terminal fragments. This mutant protein then interferes with gene transcription, particularly in the mitochondria, affecting metabolic processes and inducing oxidative stress.
Name other triplet repeat disorders thought to act by dominant GoF
SCA1,2,3,6,7,17
DM
What is the most common cause of CMT?
PMP22 duplication - a 1.5mb region
What does deletion/LoF of PMP22 cause?
HNPP
List examples of diseases showing a dominant negative effect
Marfan syndrome Non-syndromic hearing loss (Cx26/30) OI - Col1a1 and COL1A2 TP53 Alzheimer's disease (APP)
How do mutations in GBJ2/6 (Connexin 26 and 30, respecitvely) cause deafness?
Mutation in one gene prevents interaction and formation of gap junctions in the cochlear membrane (Cx26 and Cx30 form a complex together). Also involved in neurotransmitter release. As signals cannot travel through the junctions the patient is deaf.
Often AR disease, but some dominant mutations in Cx26 form a full length structurally abnormal protein that interferes with junction formation.
What are the two type of Osteogenesis Imperfecta? What is the difference in the mutation spectrum for each?
OI type 1 - less severe, caused by loss of the COL1A1 gene function through PTC and nonsense mutation. Reduced amount of functionally normal COL1A1 is available.
OI type 2, 3 and 4 - severe, caused by missense mutations affecting glycine residues. These glycine residues are instrumental in holding the collagen helix structure together. This leads to the formation of abnormal protein.
How is TP53 considered a dominant negative gene?
TP53 usually forms tetramers (2x dimers) and acts to prevent passage through the cell cycle.
Mutations in the DNA binding domain prevent TP53 complex from binding the DNA. If the dimers contain abnormal TP53, they are are functionally abnormal then they will not bind the DNA
Describe the structure and function of CFTR
2 transmembrane domains, 2 nucleotide binding domains, a regulatory domain (R) and C-terminus.
It is a cAMP activated chloride channel present in the apical membrane of secretory epithelial cells
What controls the opening/closing of the CFTR protein?
The binding of protein kinase A to the R domain.
Summarise the phenotype of CF patients.
Prenatal:
Meconium ileus, Echogenic bowel (3% of EB cases are CF)
Postnatal: FTT Digestive problems Respiratory infections Pancreatic insufficiency Chronic cough Bronchiectasis Infertility Clubbing of the fingers Sputum with staph or pseuodomonas infection.
