Lecture 21 X-Linked Disorders and Mitochondrial Inheritance

Question 1

Key feature of X Linked Recessive Inheritance

Answer 1

1. Just males effected
   -disorders caused by recessive genes, on the edges of chromosomes, proteins encoded for on X chromosome
2. Females are carriers –> pass onto sons
3. Sons dont pass onto sons
   –> often skips generations
   (like autosomal recessive disorders)

Question 2

X linked recessive inheritance

Answer 2

•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

Question 3

X chromosome linked recessive: Mother heterozygous (asymptomatic carrier) XhX

Answer 3

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)

Question 4

X chromosome linked

recessive: Father affected symptomatic XhY

Answer 4

All Daughters must be carriers

Sons will not have haemophilia (as father has to give Y in order to produce sons)

Question 5

Family History

Answer 5

Important to get an initial idea of the pedigree

Question 6

Examples of X linked recessive disorders

Answer 6

• Haemophilia
• Colour blindness
• Muscular dystrophy
• Fragile X syndrome

Question 7

Haemophilia: an example of an X linked recessive disorder

Answer 7

– 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)

Question 8

Coagulation pathway

Answer 8

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

Question 9

Clinical Manifestations of Haemophilia

Answer 9

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)

Question 10

Management of Haemophilia

Answer 10

1. 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
2. Adequate pain relief
3. Rest of affected joint

Question 11

Summary: clinical aspects of haemophilia

Answer 11

• 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

Question 12

Problems with Management of Haemophilia

Answer 12

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

Question 13

The impact of genetic technology.

Answer 13

• 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

Question 14

The diagnostic challenge in X linked recessive disorders

Answer 14

• 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

Question 15

Strategies: Pedigree Analysis

Answer 15

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)

Question 16

Strategies: phenotypic analysis clotting factor levels in blood

Answer 16

Phenotypic studies
Normal factor VIII levels 50 – 150 u/L (%)
Severe haemophilia factor levels

Question 17

Lyonisation

Answer 17

• 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

Question 18

Problems with phenotypic analysis…

Answer 18

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

Question 19

Haemophilia: genetic issues problem

Answer 19

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

Question 20

What are the molecular options for prenatal diagnosis?

Answer 20

1. Genetic Disease Analysis
   Blood, Skin, CVS, Amniotic Fluid (any DNA containing sample) –>
2. DNA (isolate and look at gene of interest)–>
   a) Polymorphic Linkage
   b) Direct mutation detection (in specific gene) (more common)
   –>
3. Diagnosis and detection of carriers (more accurate)

Question 21

Mutations causing Haemophilia

Answer 21

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

Question 22

Direct mutation analysis

Answer 22

• 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

Question 23

Indirect Genetic Analysis

Answer 23

• 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

Question 24

Linkage analysis

Answer 24

-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

Question 25

Polymorphisms

Answer 25

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

Question 26

Bcl I RFLP analysis in

Haemophilia A family

Answer 26

diaagrams

Question 27

Problems with linkage analysis

Answer 27

• 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

Question 28

Options for female confirmed as carrier (heterozygote)

Answer 28

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

Question 29

Preimplantation Diagnosis

Answer 29

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

Question 30

Therapeutic advances: Recombinants

Answer 30

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

Question 31

Therapeutic advances: Gene Therapy

Answer 31

Gene therapy strategies gene replacement therapy

• using different vectors (adenovirus to introduce virus gene and increase production of factor 8)
• The future

Question 32

Mitochondrial DNA

Answer 32

-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)

Question 33

Mitochondrial DNA (mtDNA) and disease

Answer 33

• 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

Question 34

Maternal inheritance of mtDNA genetic disorders

Answer 34

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

Question 35

Chronic progressive external ophthalmoplegia

Answer 35

• slowly progressive paralysis of extraocular muscles

* commences with ptosis