Lecture 21 X-Linked Disorders and Mitochondrial Inheritance Flashcards
Key feature of X Linked Recessive Inheritance
- Just males effected
-disorders caused by recessive genes, on the edges of chromosomes, proteins encoded for on X chromosome - Females are carriers –> pass onto sons
- Sons dont pass onto sons
–> often skips generations
(like autosomal recessive disorders)
X linked recessive inheritance
•Females (46,XXh) are carriers ie heterozygotes; usually unaffected/asymptomatic
-wildtype will produce protein
• Female carriers transmit to sons (46, XhY); 50:50 chance that son will inherit the affected/mutated Xh chromosome
•50:50 chance that daughters of female carrier will also be carriers (heterozygous)
•Affected males do NOT transmit to sons, but all daughters will be carriers
X chromosome linked recessive: Mother heterozygous (asymptomatic carrier) XhX
1 in 2 (50%) chance if each girl carrying the haemophilia gene
1 in 2 (50%) chance of each sone having haemophilia
1/4 chance of effected offspring (affected girl= asymptomatic carrier. affected son= symptomatic haemophilia)
X chromosome linked
recessive: Father affected symptomatic XhY
All Daughters must be carriers
Sons will not have haemophilia (as father has to give Y in order to produce sons)
Family History
Important to get an initial idea of the pedigree
Examples of X linked recessive disorders
- Haemophilia
- Colour blindness
- Muscular dystrophy
- Fragile X syndrome
Haemophilia: an example of an X linked recessive disorder
– X linked recessive disease
– Deficiency of coagulation factor or Functional abnormlaity of the Coagulation factor (clotting factors. results in bleeding tendency)
• Factor VIII (Hamoeophilia A)
• Factor IX (Hamoeophilia B/Christmas Disease)
-clinically and phenotypically look same, same presentation, cannot diagnose until measure in Blood Factor 8 or 9 levels
– 1 in 5000 males (not common)
-but budget for treating Heamophilia is 20-22 mil (significant effect on family and patients and health budget)
Coagulation pathway
Learn Diagram
Cut/damage
BV will constrict –> platelet clump forms –> activation of coagulation protein –> cascade –> activation of thrombinogen to thrombin –> activation of fibrinogen to fibrin
-fibrin forms clot
-most coagulation factors formed in the liver
-enzymatic cascade
FVIII FIX (factor 8 and 9) - genes for these are on X chromosome
Clinical Manifestations of Haemophilia
Recurrent and spontaneous bleeding/brusing/soft tissue bleeding following relatively minor trauma
• joints (elbow joint)
-v painful
-will result in long term repeated bleeds
-damage to normal articular surfaces
• soft tissues
• muscle
• other sites eg CNS (e.g. heamorhage into posterior cerebral hemisphere - often life threatening)
Management of Haemophilia
- Infusions of coagulation factor (replacement)
- plasma seprated off, and taken to produce specific plasma products including coagulation factos (8 and 9 made from blood products)
- most people using genetically engineers recombinant coagulation factor - Adequate pain relief
- Rest of affected joint
Summary: clinical aspects of haemophilia
• X linked recessive disorder
• Small number of patients but significant burden to patient and family:
• Physical suffering
• Ongoing need for therapy
• Risks and complications of therapy
• Social, education and work implications
• Long term complications (from transfusion) eg hepatitis B and C, HIV, possibly variant CJD, chronic joint damage
-Significant burden and disability for the families
Problems with Management of Haemophilia
Patients that had repeated transfusions of pulled blood products
- many got infected with Hep B and C
- in US paid blood donors lead to HIV infection
- UK worry for Mad Cow disease/CJD variant
The impact of genetic technology.
• Improved strategies for assessment and diagnosis of females who may be carriers
-help families make informed decisions - wherther theyre a carrier, and if they are, what options do they have re children
• Potential strategies for prenatal diagnosis (for female carriers)
• Safer therapeutic strategies
The diagnostic challenge in X linked recessive disorders
- Diagnosis of asymptomatic female carriers in a family with an X linked recessive disorder
- Offering strategies for a woman who is known carrier and wishes to have children
Strategies: Pedigree Analysis
Examine family tree give probabilities
(grandmother obligate carrier, as father is hameophiliac) (son has had haeomphilia. 1:@ chance her daughter(patient’s mother) has it ,
Therefore overall 1:4 chance that patient is a carrier
-probability (Wheather forecasting)
Strategies: phenotypic analysis clotting factor levels in blood
Phenotypic studies
Normal factor VIII levels 50 – 150 u/L (%)
Severe haemophilia factor levels
Lyonisation
• If females 46, XX; why don’t they produce twice as much protein for any X chromosome encoded gene e.g. coagulation factor VIII ? (e.g. 200)
• Due to random inactivation of one X chromosome (maternal or paternal) in somatic cells; (factor 8 levels will be normal in a normal female)
(factor 8 in a carrier (1 mutant one wildtype) random inactivation, half liver cells wont make and half cells will)
inactive X = Barr body; occurs early at approx day 16
Problems with phenotypic analysis…
Variation in normal clotting factor levels e.g. with exercise, stress, using estrogens etc
-will increase factor 8 levels
-lots of environmental things influence the levels
-give clue, but approx 20% error, due to overlap betwee normal rangeand range seen in carrier
Requires fetal blood sample for prenatal diagnosis, therefore late and difficult
-could confirm mother as carrier, but couldn’t test pregnancy for haemophilia
-would have to get fetal blood sample which is
1)technically difficult
2) can only be done late in pregnancy
–>therefore doesnt lend well to genetic prenatal diagnostic strategies
Haemophilia: genetic issues problem
21 year old
wants to have children
family history of heamophilia
maternal uncle had haemophilia
-take family history
-unlikely to be new mutation
-grandmother is an obligate carrier, therefore 50% chance that mother was carrier
-then if mother was carrier then 50% chance that your patient is a carrier
-what might her being a carrier mean
-this question wouldn’t be to a specialist. might be to a mid wife, house officer, obstetritian
1. Am i carrier
2. If i am a carrier, i have seen the disability/effects it has had on my uncle, i am not sure whether i am able to bring up a child with heamophillia
-what options do we have
What are the molecular options for prenatal diagnosis?
- Genetic Disease Analysis
Blood, Skin, CVS, Amniotic Fluid (any DNA containing sample) –> - DNA (isolate and look at gene of interest)–>
a) Polymorphic Linkage
b) Direct mutation detection (in specific gene) (more common)
–> - Diagnosis and detection of carriers (more accurate)
Mutations causing Haemophilia
Mutation in genes of Factor VIII or FIX • Structural changes eg deletions • Point mutations Factor VIII gene -massively complex -in hameophilia and many other genetic diseases there is a whole spectrum of mutations differing between patients
Direct mutation analysis
• Analyze DNA from affected patient – identify mutation
-could use index case, to see if that mutation is present
• Potential female carriers eg sister of a haemophilia patient – can accurately confirm carrier status
-often just providing reassurance, proceed to pregnancy
-99% of family
Indirect Genetic Analysis
• Direct mutation analysis preferred strategy
• If mutation in family not known can use linked polymorphic markers / SNP’s to track the abnormal gene in a family
-polymorphic markers / SNP’s are normal variations in DNA, in noncoding regions of genes. Single base pair changes and no-coding regions inherited from parents non pathogenic. Used to track an abnormal gene through family
-becoming rarer and rarer with developing technologies and sequencing
Linkage analysis
-not looking for disease causing mutation
-just using the normal variations that occur within the DNA to track a gene (polymorphic markers / SNP’s)
-polymorphisms (single base changes) Change restriction enzyme sites (made by bacteria and cut DNA at particular sequences)
-if the sequence is changed may result in loss or addition of a cutting site = different sized DNA fragments
• RFLPs
Linkage analysis
– (restriction fragment length polymorphisms
• VNTRs
– variable number tandem repeats
-DNA fingerprints, use double or triplet repeat, number of which are inherited by parents, used to track normal Gene
Polymorphisms
Base changes have changed the enzymes
-use enzymes to cut the “meant” length of DNA with the enzyme.
run through gel to separate by size.
hybridize to labelled probe
Bcl I RFLP analysis in
Haemophilia A family
diaagrams
Problems with linkage analysis
• Requirement for family studies including availability of index case
• Non-paternity (sometimes father isnt the father)
• Recombination
• Lack of an informative marker / polymorphism
-homozygous mother
-wouldnt know which is which
–> therefore preference is direct mutation analysis
Options for female confirmed as carrier (heterozygote)
Mutation analysis of fetal DNA obtained by -chorionic villous biopsy or -amniocentesis -do direct mutation anaylsis -reassurance if confirm normal pregnancy -if tissue is effected and hence fetus is effected provides family with options (termination or prepare for delivery of hemophilia child - nescessary support re management and delivery) -SAME DIRECT mtuation anaylsis RARE linked anaylsis
Preimplantation Diagnosis
In vitro fertilization followed by analysis of 6-8 cell blastocyst:
-pipette off single cell without affecting embryo viability
– Select non male embryos
– positively Select embryos with normal factor VIII gene
Therapeutic advances: Recombinants
Recombinant factor VIII and IX
– Removes risk of transfusion transmitted infection, safer, home therapy, prophylaxis
-treatment for almost all new/recently diagnosed haemophilia patients
-recombinants = genetically derived (gene has been cloned)
-products produced for a lot of enzyme deficiencies
Therapeutic advances: Gene Therapy
Gene therapy strategies gene replacement therapy
- using different vectors (adenovirus to introduce virus gene and increase production of factor 8)
- The future
Mitochondrial DNA
-very rare (wont see these patients unless really specialised)
-useful research tool
• Double stranded circular DNA
• MATERNALLY inherited (from mother)
• 16,569 nucleotides
• 37 genes:
encode 13 proteins (oxidative phosphorylation system), 22 tRNAs, 2 rRNAs lack of non-coding (intron) sequences
-the mutations seen tend to be in high oxygen/energy use body organs (brain, eye, cardiac and skeletal muscle)
• Each mitochondria has 2 to 10 DNA molecules
• Each cell contains multiple mitochondria
• Mutation rate is 10x that of nuclear DNA
does not have protective histones or effective repair mechanisms (less effective repair mechanisms)
Mitochondrial DNA (mtDNA) and disease
• Mutations in mtDNA produce effect by deficiency in respiratory chain (5 enzyme complexes within inner mitochondrial membrane)
-oxidative phosphorylation and ROS management
-production of ATP
-apoptosis
-production of reactive oxygen -species cellular oxidation and reduction
• Each of respiratory chain complexes made up of protein subunits encoded by both nuclear DNA and mt DNA
• Diseases associated with mtDNA mutations affect organs with high energy requirements
-eye
-brain
-skeletal and heart muscle
Maternal inheritance of mtDNA genetic disorders
All inherited through the MOTHER
- although males are affected, it is all inherited from the mother
- Pedigree of a kindred transmitting a mitochondrial mutation causing the syndrome of myoclonic epilepsy with ragged red fibers (MERRF)
- irregular contour of muscle fibres due to subsarcolemmal collections of mitochondria that appear red with trichrome stain
Chronic progressive external ophthalmoplegia
- slowly progressive paralysis of extraocular muscles
* commences with ptosis
