Molecular Genetics – Core Constitutional Diseases And Cancer Studies Core constitutional diseases and Cancer services Flashcards
(27 cards)
What is the phenotype seen in those with cystic fibrosis?
A disorder affecting the respiratory tract and digestive system:
- Pancreatic insufficiency; Meconium ileus
- Pulmonary infections, obstructive lung disease
- Male infertility – azoospermia, CBAVD
CFTR-related disorders:
- CBAVD; Idiopathic pancreatitis; bronchiectasis
Multi organ disorder. Pancreatic enzymes are prematurely activated before reaching duodenum. Means sufferers may need enzymes to help with digestion as the enzymes aren’t present in the duodenum.
What is the genotype of cystic fibrosis?
Classic autosomal recessive inheritance, incidence of 1/2500 in English Caucasian.
Carrier frequency is 1/25
Most common mutation is in Phe508del.
What is the pathogenicity of CF, and so what is the test performed?
Cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel with 12 transmembrane domains, that regulates chloride ion transport (pumps out Cl, but inhibits inner sodium transport).
This process creates the optimum osmotic conditions; water flows from inside to outside of apical membrane.
This water lubricates secretions, so they have a low enough viscosity.
Due to mutations, the action of these channels is impaired in CF so water doesn’t flow in the same volumes out of the cell, this makes secretions very viscous.
Due to reduced water efflux there is a higher salt concentration on the outside of the cell = WHY THE SWEAT TEST IS THE GOLD STD FOR CF. Sweating in induced in babies and young kids is induced, gives a NaCl reading of around 30-40 mmol/U - outside normal range (CF range is 80-90).
Which genes and proteins are affected in CF?
Gene – Cystic fibrosis transmembrane conductance regulator (CFTR)
Chromosome location - Chromosome 7
Protein function
- ATP binding cassette superfamily.
- Chloride channel for secretary endothelial cells
- Transports Cl ions across the epithelial surfaces – to help control the
movement of water and regulate mucus consistency.
Where do referrals and diagnoses for CF come from?
Referrals
Diagnosis/Confirmation of diagnosis
- Meconium ileus/failure to thrive
- Sweat test raised
- Bronchiectasis
- CBAVD
Carrier testing
- Family member
- Partner - because it is a recessive disease
Echogenic bowel - association with CF (11-13%)
Egg/Sperm donor screening - people going for IVF
How is CF mutation testing performed?
Routine (first tier) testing involves testing for around 50 common mutations -Elucigene Kit (EU2v1) - add-ons that target specific ethnic groups.
Most common mutation is p.(Phe508del) in CF populations worldwide but frequencies of different CF mutations vary in different populations. eg. p.(Phe508del) represents 75.3% of mutations in UK but only 24.5% in Turkey.
2nd Tier service :Screen whole gene but NOT a core service -Manchester offers this extended screen by NGS and MLPA (dosage or copy number) dosage.
What role does the PolyT Tract have in CF?
The intron 8 poly-thymidine (PolyT) repeat affects the efficiency of the exon 9 splice acceptor site. PolyTG tract (directly 5’ to PolyT) There are three alleles (9T>7T and >5T) that are all different lengths and therefore all affect splicing to different degrees. The longer the PolyT Tract, the more efficient splicing is. Short PolyT results in a splicing defect that involves exon 9 being skipped and missed out of the resulting mRNA with disease consequences. The severity of the phenotype depends on the length of the PolyT; a 9T/9T genotype would produce ~100% normal CFTR mRNA (ie includes exon 9), whereas a 5T/5T genotype produce ~10%
What is the phenotype of Duchenne Muscular Distrophy?
Muscle degeneration leads to difficultly walking and breathing
Cardiomyopathy
Onset <5 years, life expectancy about 25 years
About 20% female carriers have mild symptoms
BMD (milder relative of the disease) – some functional dystrophin
Which genes and proteins are effected in DMD?
Gene – Dystrophin gene - X-linked so predominates in males. Protein is a structural component in skeletal, muscle
Chromosome location – Xp21
Protein functions as a structural component within muscle tissue. It provides structural stability to dystroglycan complex on cell membrane. Distophin binds to actin filaments to perform a stabilising role in the membrane complex, which protects muscle fibres from their long term muscle contraction-relaxation cycles. Evidence that the protein complex is involving lives in cell signalling as mutations leads to apoptosis.
Absence of dystrophin protein results in muscle degeneration and release of creatine kinase (used as a diagnostic indicator).
What is the mode of inheritance of DMD?
Inheritance – X-linked - males carrying a mutation will be affected. Females with one will be carriers but the distribution of the mutation on the active vs non-active chromosome will determine whether they have symptoms = manifesting carrier.
Incidence 1 in 3600 males
1/3 DMD are de novo, about 10% of these are due to gonadal mosaicism. females may be unaffected in the blood, but have 10 % chance of being mosaic in uicytes
Where do referrals for DMD come from?
Referrals
- Confirmation of clinical diagnosis
- Carrier testing - very important with this disease
- Prenatal diagnosis
What are the tests used to diagnose DMD?
IHC for dystrophin on muscle biopsies - lack of indicates DMD
Creatine Kinase levels in blood are elevated
Electromyography – shows muscle tissue weakness
Molecular genetic testing
- MLPA (multiplex ligation probe amplification) – about 65%
mutations are intragenic deletions; 5% are duplications (deletions or
multiplications of single or sequential exons)
- Frameshift hypothesis
Screening – sanger sequencing (79 exons 2-4Mb)
What is DMD/BMD mutation testing?
Dosage analysis using a commercial kit:MLPA (multiplex ligation-probe amplification). Uses 2 separate probe sets: P034 and P035.
Using a series of probes, each exon in the gene has a probe that binds in proportion to the number of copies of that exon you have. Then amplify it up using PCR and a bound probe, so the signal you get is proportional to the number of exons. So if you have a deletion then in the case of a male there will be no signal at all, in a female you will only have half the signal.
When does the genotype lead to DMD and when does it lead to BMD? How would testing reveal which using molecular testing?
Whether you have DMD or BMD is determined by the type of mutation you have, or rather the end point of the mutation.
If the deletion is in frame - it encapsulates codons and doesn’t interrupt codons, then you will have BMD (discrete number of codons, therefore AA that are disrupted).
If it it interrupts the middle of a codon it leads to a frameshift mutation (frame of triplet codon is altered, therefore AA), this leads to a nonsense mutation = more severe and leads to DMD.
There are exceptions, for example deletions at the start of the reading frame, eg exon deletion of 3-7, gives you an intermediate variable phenotype - difficult to predict phenotype and need studies like BIHC to see if there’s any dystrophin present. Microarray can also be used to do this (genomic scan of copy number variants across the genome) can show copy number variations in dystrophin.
What are trinucleotide repeat disorders?
First described in 1991
- Fragile X syndrome (CGG repeat)
- Huntington disease
- Spinal and bulbar muscular atrophy (CAG repeat)
Over 40 disorders described to date
- Neurological
- Neurodegenerative
- Neuromuscular
Inheritance usually autosomal dominant (only need to inherit one affected chromosome)
- Exceptions include FRAXA/FRAXE – X-linked and FRDA – autosomal
recessive
Tend to be a triplet of bases (perhaps more) that are unstable leading to expansion - different run of these repeats. Once they pass a threshold you get a disease. If the expansion increases over generations (e.g. Huntington’s and FraX this is called anticipation)
What is the phenotype of fragile X Syndrome?
Mental retardation – moderate to severe and social impairment
Large ears, long face
Variable severity – mosacism/ skewed X inactivation
Fragile X Ataxia Tremor Syndrome (FXTAS)
Premature ovarian failure (POF) - predominantly affects a discrete expansion range
What genotype causes fragile X and what is the molecular pathology?
Gene – FMR1 - role is to suppress translation
Chromosome location – Xq27.3 Fragile site A
Protein is thought to be a suppressor of mRNA translation via binding non coding RNA.
Molecular Pathology
Triplet repeat in 5’UTR – CGG
Full expansion causes methylation of the promoter and transcriptional silencing, thus loss of protein function. But smaller expansions, eg 59 repeats lead to premutations.
Premutations – no methylation, no loss of function.
RNA gain of function – expanded mRNA sequesters proteins (eg CUGBP1) involved in transcription - unknown how this leads to disease.
MALES WITH A PRE-MUTATION WILL NOT GIVE RISE TO CHILDREN WITH A FULL MUTATION AS THE SPERMATOZOA WILL NOT EXPAND TO FULL MUTATIONS - not viable
ONLY FEMALES CAN TRANSMIT A PREMUTATION INTO A FULL MUTATION
How do you test for fragile X?
Standard, Long-range and TP-PCR:
CGR repeat in FMR1 gene. TP-PCR is a commercial kit with an advantage over standard PCR as it can expand ~200 repeats.
Two tests in one as you also have a triplet primer that binds to multiple locations in one repeat, bind and expands PCR from multiple locations.
What are the different classes of cancer genetics?
Inherited (Germline) cancers – breast /ovarian cancer BRCA1/2, Lynch syndrome MSH2, MLH1, MSH6 (PMS2),
Familial adenomatous polyposis (FAP) APC,
Neurofibromatosis 1 and 2 NF1, NF2
- all are tumour supressor genes
Somatically-acquired mutations – activating mutations –drugable targets.
Not loss of function but gain, switch proteins on and permenantly activate receptors - lose regulation
What is Knudson’s two hit hypothesis in tumour suppressor genes?
Refers to familial cancers. A “two-hit” model - If you have one mutation you’re more likely to get a second one.
How is Hereditary Breast/Ovarian Cancer associated with the BRCA genes?
Suspected in individuals with personal or family history of the following:
- Early onset breast cancer (or male breast cancer)
- Ovarian cancer (or both breast and ovarian)
- Triple negative breast cancer
- Pancreatic cancer with breast or ovarian in the same individual or
on the same side of the family
- Ashkenazi Jewish ancestry
HBOC diagnosis confirmed with identification of germline mutation in BRCA1 or BRCA2
Chromosome location – Chr17/Chr13
Protein function:
BRCA1: Tumour suppressor gene involved in cell cycle progression, gene transcription regulation, DNA damage response and ubiquitination. Co-localises with BRCA2 and RAD51 at site of DNA damage.
BRCA2: Tumour suppressor gene with no homology with BRCA1. Interacts with RAD51 and is involved in DNA damage repair via homologous recombination.
What is the molecular pathology of Hereditary Breast/Ovarian Cancer?
Tumour suppressors genes involved in Homologous recombination - use up damaged strand to repair the damage.So loss of function impairs this.
Error in DNA damage repair mechanism
What is the inheritance pattern of H ereditary Breast/Ovarian Cancer?
Inheritance – Autosomal Dominant
Risk of developing breast cancer is up to 90% if BRCA1 and up to 85% if BRCA2 by age 80
Risk of developing Ovarian cancer (lifetime risk) is up to 40-60% if BRCA1 and up to 10-30% if BRCA2
What is the assay design for BRCA 1 and 2 testing?
Target enrichment method: long range PCR
Amplicons generated from the region of interest
i.e. BRCA1/2
Coding exons grouped into a minimum number of potential amplicons (reduce unnecessary intronic content)
BRCA 1 and 2 amplified in 22 long fragments between 2 and 7 kB
