Test 2 Flashcards
Monogenic definition
strong genetic influence by 1
gene
Recessive definition
Need two defective copies to be
affected
Cystic fibrosis is due to:
mutations in the gene for the CF transmembrane conductance regulator
(CFTR) gene, Cl- channel
Characteristics of cystic fibrosis:
- fatal monogenic recessive disease
- congenital (present at birth)
- highest frequency in Northen Europe
- Heterozygote/carrier frequency of 1/20
- General frequency in Caucasian populations: 1/1600-3000
- less common in African, Arab or Asian populations
Inheritance pattern of CF with two carriers as parents:
- 25% offspring unaffected, 50% carriers (unaffected), 25% have the disease
Inheritance pattern of CF with one normal parent and one parent as a carrier
- 50% of offspring are carriers (unaffected), 50% of offspring do not carry the mutation at all (unaffected)
Phenotype/clinical presentation of CF:
– increased sweat Na and Cl
– pancreatic insufficiency
– pulmonary infections
- thick mucous production
- 90% of males are infetile - congenital bilateral absence of the vas deferens
- > 90% of deaths due to recurrent infection, most commonly with Pseudomonas aeruginosa
Root of pathophisology of CF
Stems from a defect in CFTR, a
chloride transporter on cell
membrane
Pathophysiology of CF
- less chloride channel activity
- change in regulation of epithelial sodium channel
- increased sodium, increased water absorption, decreased water content of secretions (also increased sodium in sweat)
- mucous becomes thicker -> impaired flow, plugs and obstructions
Effect of pathophysiology of CF on GIT
- Blockage of pancreatic ducts.
- Loss of water from GIT
- Meconium ileus
- Constipation
- Rectal prolapse
Effect of pathophysiology of CF on lungs
Decreased periciliary volume + thick
mucus leads to poor ciliary clearance of pathogens and recurrent infections
CFTR gene
- Chromosome 7q31-32 - 250Kb region
- 27 exons, 6.5Kb transcript
- Regulated by cAMP sensitive protein kinase
CFTR protein
- A 1480 aa membrane protein.
- A Cl- ion channel expressed in the apical membrane of exocrine
epithelial cells. - Member of the ATP binding cassette family of transporters
Mutations in CTFR
- > 1,000 mutations described.
- One major mutation in Caucasians, ΔF508, accounts for ~ 70%
- A few at frequency of 1-3%; the rest at very low frequency.
Classic CF clinical phenotype is associated with:
- Pancreatic exocrine insufficiency (highest correlation between genotype and phenotype)
- COPD (lowest correlation between genotype and phenotype for lung manifestations)
- Abnormal concentrations of sweat electrolytes
- No vas deferens in males
- Bowel obstruction
Pulmonary manifestations in genotype/phenotype correlations (CF)
- Most common cause of morbidity and mortality.
- But there is a poor correlation with severity of genotype and the age on
onset, severity and progression. - For example, even among homozygotes for the most common mutation
(DF508), lung function may vary from normal to severe dysfunction
How heterozygous CF patients have an advantage against diarrhoea
- in normal patients: toxins released by cholera and E coli act on CFTR, cause increased fluid flow in intestine –> diarrhoea
- CF homozygotes don’t secrete chloride ions in response to bacteria
- Mutations can protect against diarrhoea
How heterozygous CF patients have an advantage against typhoid fever
- Salmonella typhi enter epithelial cells via interactions with CFTR.
- One study, showed an 86% reduction in internalization of S. typhi into the gastrointestinal tract, of mice
heterozygous for Cftr-ΔF508, relative to that in wild-type mice - Thus, selection for typhoid fever resistance provides another possible
explanation for the high frequency of CFTR mutations
Newborn screening for CF
- Measure immunoreactive trypsinogen (IRT), 48-72h after birth (these levels are raised in CF), blood is from heel prick
- Genotyping: two mutations = CF, no mutations = unlikely to have CF, one mutation = carry out sweat test to determine electrolytes
- CF patients cannot absorb NaCl from sweat
Sweat Test Interpretation for CF
- Australian recommended values for sweat chloride in infants in newborn screening (should be done after 1st postnatal week):
- Cl 60 mmol/l, cystic fibrosis;
- Cl 30–59 mmol/l, borderline;
- Cl 29 mmol/l, normal.
- Up to 25% of infants with MI do not have an IRT value above the cutoff level. Any neonate with a family history of CF or Meconium Ileus SHOULD be followed up
Fragile X syndrome:
- most common inherited intellectual disability
- can range from learning disabilities to severe cognitive disabilities to intellectual disabilities
- 30% of individuals with Fragile X have autism
- 2-6% of individuals with autism have Fragile X
- diagnosed with bloods or DNA testing
Prevalence of Fragile X
- Affects 1/4000 males and 1/6000-8000 females
- Can appear in all socio-economic backgrounds
- 50% of females with full mutations have some form of ID
Clinical features of Fragile X
- Large ears, long narrow face w/ prominent forehead, mitral valve prolapse, seizures, eye problems
- FGX mutations affecting brain development - mental impairment, developmental delays, learning disabilities
Cause of Fragile X disorder
- more than 200 repeats in CGG expansion
- this leads to hypermethylation of cytosine residues
- causes deactivation of FMR1 gene (no FMRP produced in Fragile X)
Analysis of mutations of Fragile X
- Stable: <45 repeats, unmethylated, individuals not affected
- Grey zone: 45-54 repeats, unmethylated, individuals not affected
- Pre-mutation: 55-200 repeats, unmethylated, individuals usually not affected
- Full mutation: >200 repeats, completely methylated, 50% of women and 100% of men affected
Fragile X associated primary ovarian insufficiency
- carriers of FX pre-mutation (55-200 CGG repeats)
- produce more mRNA but less FMRP protein
- in females, menopause is 5 years earlier and 23% of female individuals have ovarian insufficiency
- toxic RNA gain is said to be responsible
Fragile X associated tremor ataxia syndrome
- affects older adults that carry pre-mutation (increased rate with age, increased rate of hypertension)
- intention tremor and cerebellar gait ataxia are main clinical features
- affects 40% of male carriers and 8-16% female carriers
FMR1 gene
- chromosome Xq27
- CGG repeats happen in 5’-UTR (healthy individuals have 6-55 copies)
- methylation of CpG site -> silencing gene -> deficiency in FMRP protein and intellectual disability
Lab testing for Fragile X
- PCR, Southern Blot -> no. of CGG expansions
- Microarray and MLPA to detect deletions
- Next Gen sequencing
Fragile X treatment
- Glutamate antagonists - reduce activity of metabotropic receptor
- Lithium - improved behaviour but not cognition
- MMP9 - reversal of structural damage in neurons
- GABA agonists (arbaclofen) - alleviate anxiety and learning disabilities
Clinical features of Huntington’s disease
- Progressive neurodegenerative disease leading to dementia
- Symptoms include mood and character changes, defects in memory and attention, progressing to movement disorders
- Onset is usually at 35-55 years and symptoms evolve over 12-15 years
Genotype/phenotype of Huntington’s
- Autosomal dominant
- Instability of CAG repeat length in sperm and oocyte DNA
- A parent that has Huntington’s (heterozygous) has a 50% chance of passing it to their children if they are with an unaffected partner
Genomic information HD gene (Huntington’s)
- On chromosome 4p16
- has 67 exons
- protein product ins huntingtin
- mRNA codes for 3144 amino acids
Errors in HD gene (Huntington’s)
- repeat region of CAG (197-265bp)
- codes for glutamine
- 23 glutamines in repeating sequence of the normal protein
HD assay (Huntington’s)
- Determine CAG repeat sequence
- Use sizing standards between 27-40 repeats
- Participate in proficiency testing
- Resolve homozygous alleles
Huntington’s genome guidelines:
- Normal allele: <26 CAG repeats, normal phenotype
- Mutable normal allele: 27-35 CAG repeats, normal phenotype (risk that children will develop it)
- HD allele (1): 36-39 CAG repeats, HD phenotype but may not develop it
- HD allele (2): >40 CAG repeats, HD phenotype
Genetic testing for huntingtons
- predicitive testing - future onset
- diagnostic testing - those who develop symptoms
- Prenatal: CVS-DNA, direct gene analysis, exclusion testing
Pathology (Huntington’s)
- exact function of Huntingtin protein is unknown, but accumulation of abnormal protein can cause neurological changes and interferes with neurotransmitters
Possible mechanism of action (Huntington’s)
- Huntintin widely expressed in cytoplasm
- amino-terminal fragments of mutant huntingtin aggregate in nuclear and cytoplasmic inclusions and destruct neurons
- primary cause: soluble or aggregated form of mutant Huntingtin
Huntington’s disease can also be involved with:
- breast cancer
- hereditary haemachromatosis
- familial hypercholesterolaemia
Principles of predicitive testing (Huntington’s):
- information collected, at risk individual is identified
- accurate risk information is given
- implications are explored and patient is provided with a time out to consider
- further counselling and details of results session
- informed consent to collect samples
- results and follow up
Five domains to consider when diagnosing Huntington’s
- Occupational
- Fiscal
- Activities of daily living
- Household chores
- Residence
Forensics in the past
- ABO blood groups to identify people
- Not sensitive, lots of biomaterial
- Low discrimination, exclusionary, supporting evidence
- Cannot ID unknown suspects, no databasing
Forensics now
- DNA to identify people
- Very sensitive, invisible traces, many crimes/cold case
- High discrimination, evidentiary, primary evidence
- Can ID unknown suspects, amenable to databasing
Services that the forensics lab provide:
- DNA analysis (criminal, DVI, parentage, missing persons, databases)
- Hair analysis, animal species identification, stain identification
- Fabric damage assessment, blood stain pattern analysis
Types of offences - serious crime
- homicides
- sexual assaults
- armed robberies
- assault/wounding
- drug possession or manufacture
- criminal parentage
TYpes of offences - volume crime
- burglary
- break and enter
- motor vehicle theft
Types of offences - other
- ID of unknown deceased
- missing persons
- disaster victim identification (natural disaster, accident, criminal action)
Stages of DNA profiling:
- Extraction
- Quantitation
- Amplification
- Analysis
DNA Extraction (stages of DNA profiling)
- various methods: manual and automated
- inhibitory substances common
- complex substrates
- liquid handling robotics
DNA Quantitation (stages of DNA profiling)
- Real-time PCR
- Human-specific
Amplification (stages of DNA profiling)
- Multiplex PCR
- thermal cyclers
Analysis (stages of DNA profiling)
- Capillary Electrophoresis (rapid, accurate, sensitive)
Short tandem repeats
- high heterozygosity (differentiate people)
- regular repeat unit (predictable alleles)
- distinguishable alleles
- robust amplification
- can be multiplexed (less DNA required, faster)
- small product sizes (<500 bp range) are better for degraded DNA
ABO blood typing vs Forensic DNA profiling
- A blood group can be present in 47% of the population, and is better at excluding people rather than including people
- The chance of two unrelated people have the same DNA profile is less than 1 in 100 billion, and the world population is around 7.7 billion –> good at individualising people
When DNA profile statistics don’t matter
- At trial, it usually doesn’t matter whose DNA it is, it matters how it got there
What you can and can’t determine from a forensic DNA profile
- you can determine the gender of the individual
- can’t determine ethnicity, racial background, age, physical appearance or illnesses
Negative results in a forensics laboratory
- you can only comment on the findings in the laboratory, not if an event had occured or not
- sperm is never detected in 50% of sexual assault cases
DNA Databases state and national
WA: Criminal Investigation (made July 2002)
National: NCIDD (National Criminal Investigation DNA Database), ACIC (Australian Criminal Intelligence Commission)
Markers used in fornesic biology
- ABO blood groups: low discrimination, fast analysis
- RFLP multi locus probes: high discrimination, slow analysis
- Multiplex STRs: high discrimination and fast analysis
What STRs and SNPs detect in Next Gen and Massively Parallel
The identity of the individual:
- Global autosomal STRs, Y-STRs, X-STRs, Identity SNPs
What they look like:
- Phenotypic SNPs
Where they are from:
- Biogeographical Ancestry SNPs
Examples of some genealogy sites
- LivingDNA
- MyHeritage
- FamilySearch
- Ancestry
- Family Tree DNA
- 23 and Me
Myths - the CSI effect
- There is always forensic evidence
- Evidence is easy to locate
- Evidence is easy and quick to test
- Evidence always provides simple, clear-cut answers
- Cases are always solved quickly
Intrinsic pathway to common pathway of coagulation
- XII converts to XIIa
- XIIa converts XI to XIa
- XIa converts IX to IXa, which binds to VII and protein C
- Compound allows conversion of X to Xa, which binds to Va
- This compound converts prothrombin (II) to thrombin
- thrombin converts fibrinogen (I) to fibrin
Extrinsic pathway to common pathway of coagulation
- tissue factor (III) binds to VIIa
- compund activates conversion of X to Xa, which binds to Va
- This compound converts prothrombin (II) to thrombin
- thrombin converts fibrinogen (I) to fibrin
Factor V East Texas severity:
- a moderately severe
bleeding disorder
Factor V East Texas symptoms:
- Easy bruising
- Epistaxis
- Bleeding after trauma or surgery
- Menorrhagia
Lab characteristics Factor V East Texas
- prolonged PT and aPTT
- Normal factor levels
- Mixing studies do not suggest presence of an inhibitor
- Patient is not on a pharmacological inhibitor
- [Platelet] = Normal
Hypothesised principle of FV East Texas
- novel mutation of F5
- A→G in Exon 13 F5
- Ser→Gly at a.a. 756
- [FV] is normal in affected individuals
Location of F5 mutation
- AA 756 is in the B domain
- Only the Heavy and Light Chains are required for factor V procoagulant activity
- The B-domain is not required for factor V procoagulant activity
Cleavage of Factor V
- Factor V is activated by thrombin or factor Xa. Thrombin cleaves the B-domain at aa’s 709, 1018, and 1545.
- Factor Xa cleaves the B-domain at aa’s 709 and 1018, but is very inefficient at
cleaving at aa 1545 - Both proteins separate the basic region from the acidic region, producing an active form of factor V
- Factor Xa is in the B domain near the acidic region, thrombin is in the heavy chain
Function of TFPI
- TFPI inhibits fXa and TF:fVIIa
- At normal physiological concentrations, TFPI must bind to fXa before it can inhibit TF:fVIIa
- At elevated concentrations, TFPI can inhibit TF:fVIIa
independently of fXa - TFPI binds to forms of fVa that retain the acidic region
How TFPIa binds FV
- Basic region of TFPIα binds to acidic region of fV - the basic regions of each factor are homologous
Exon 13 FV East Texas
- Exon 13 is shortened
- PCR performed using F primer in exon 12 and R primer in exon 14
- Unaffected - complete exon 13 transcript (top) is bright
- Affected: novel exon 13 transcript (bottom) is bright
How exon 13 is shrotedin FVET
- The A→G mutation produces a novel splicing donor site that causes an in-frame deletion of 702 aa
Xa- activated Factor Va vs fV-Short in FVET
- Xa- activated Factor V - B domain is from aa 1019 to 1545
- fV-Short - B domain is from aa 1458 to 1545
Calibrated automated thrombography FV East Texas
- measures thrombin generation in plasma
- plasma incubated with low TF concentration
- Coagulation is initiated with the addition of Ca2+
- Thrombin generation is lower in FV East Texas patients
Relationship between TFPIa and TG
↑ Plasma TFPIα is the cause of ↓ TG
What type of mutation is FVET
indirect, gain of function
mutation
Individuals with Fv Amsterdam:
- have ↓ TG that is corrected by anti-TFPI antibody
- have a shortened fV protein
- due to a novel mutation in exon 13 of FV with deletion of aa 623 in the B domain (basic region removed)
