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READ:Gelehrter et al. pages 137-143SEE: Course web site, lecture notes

Bleeding disorders have been recognized since ancient times…• The Talmud (2nd century AD) states that male babies do not have to be circumcised if two brothers have died from the procedure

• In 12th century Albucasis, an Arab physician, wrote about a family in which males died of excessive bleeding from minor injuries

• In 1803, Dr. John Otto, Philadelphia, wrote about an inherited hemorrhagic disposition affecting males

• In 1828 at the University of Zurich, “hemophilia" was first used to describe a bleeding disorder

platelet plug

fibrin clot

vasoconstriction

vascular injury

incompleteplatelet plug

incomplete or delayedfibrin clot

vasoconstriction

vascular injury

NORMAL BLEEDING DISORDER

A “Royal Disease”

Queen Victoria (1837 to 1901) passed hemophilia on to German, Russian and Spanish royal families. Her son, Leopold, had frequent hemorrhages (British Medical Journal,1868) and died of a brain hemorrhage at 31. His grandson also died of a brain hemorrhage in 1928.

extrinsic pathwayintrinsic pathway

Types of Bleeding Disorders

von Willebrand disease Factor I deficiencyFactor II deficiencyFactor V deficiency Factor VII deficiencyFactor VIII deficiency (Hemophilia A) Factor IX deficiency (Hemophilia B)Factor X deficiency Factor XI deficiency Factor XII deficiencyFactor XIII deficiency

Molecular Genetics of HemophiliaMolecular Genetics of Hemophilia

• Hemophilia A Hemophilia A – Factor VIII deficiencyFactor VIII deficiency

• Other Genetic Disorders with low Factor VIIIOther Genetic Disorders with low Factor VIII– Von Willebrand Disease (Type 2N)Von Willebrand Disease (Type 2N)– Combined Deficiency of Factor V and Factor VIIICombined Deficiency of Factor V and Factor VIII

• Hemophilia BHemophilia B– Factor IX deficiencyFactor IX deficiency


Hemophilia AHemophilia A• Incidence 1:5,000 - 1:10,000 malesIncidence 1:5,000 - 1:10,000 males

– about as rare as the birth of tripletsabout as rare as the birth of triplets

– ~ 1 in 5,000 live male births are affected. ~ 1 in 5,000 live male births are affected.

– ~ 15,000 to 20,000 people with hemophilia in the US~ 15,000 to 20,000 people with hemophilia in the US

• Hemarthroses, post-traumatic and post-surgical bleedingHemarthroses, post-traumatic and post-surgical bleeding

• Severity related to factor VIII levelSeverity related to factor VIII level– <1% = severe<1% = severe

– 1-5% = moderate1-5% = moderate

– 5-15% = mild5-15% = mild

• Inhibitors develop in ~10-20% of severe patientsInhibitors develop in ~10-20% of severe patients

Symptoms of hemophilia include...

Primary: Bruising and Bleeding

Minor bleeds:• early joint and muscle bleeds• bleeding in the mouth and gums• epistaxis (nosebleed), • hematuria (blood in the urine)

Major bleeds • central nervous system• severe injury• neck/throat, eye, gastrointestinal, hip, iliopsoas, late joint and

muscle, testicles, and retroperitoneum bleeds

Secondary:

• Chronic joint deformities from recurrent bleeding • Antibodies to transfused factor VIII (inhibitors develop only in 20-30% of severe patients, not in mild-moderate) • AIDS - Over 60 %of persons with hemophilia treated with plasma concentrates in the early 1980s became HIV+

Mild hemophilia patients (factor levels >5% and <50%) - usually bleed only after injury or surgery- some never have a major bleed, others have several

episodes depending on functional factor levels- carriers of hemophilia may fall in the mild range

Moderate hemophilia patients (factor levels 2% to 5%) - bleed about one a month, usually after trauma,

surgery, or exertion. - once a bleeding history is established in an area, may have

spontaneous bleeding episodes into those areas

Severe hemophilia patients (factor levels <1%) - bleed very easily, sometimes spontaneously with no

warning and for no apparent reason, usually targeting the joints but potentially in any area

Hemophilia A: GeneticsHemophilia A: Genetics• X-linked inheritanceX-linked inheritance

– ~1/3 patients represent new mutations (Haldane hypothesis)~1/3 patients represent new mutations (Haldane hypothesis)

• Germinal mosaicismGerminal mosaicism

• Low FVIII in female consider:Low FVIII in female consider:– skewed X-inactivationskewed X-inactivation

– chromosomal abnormality (normal X inactivated)chromosomal abnormality (normal X inactivated)

– VWD (particularly type 2N)VWD (particularly type 2N)

X-Linked Recessive InheritanceX-Linked Recessive Inheritance

Carrier femaleCarrier female

Affected maleAffected male

Normal maleNormal male

• Affected males (XY):Affected males (XY):–sons unaffected (no male to male transmission)sons unaffected (no male to male transmission)–daughters obligate carriersdaughters obligate carriers

• Carrier female (XX): Carrier female (XX): –½ sons affected; ½ daughters carriers½ sons affected; ½ daughters carriers

• Affected females: very rare.Affected females: very rare.

New mutation in New mutation in germ cellgerm cell

New mutation in New mutation in maternal or maternal or

paternal germ cellpaternal germ cell

Is this woman a hemophilia carrier of hemophilia? • A biological daughter of a man with hemophilia• A biological mother of one son with hemophilia • A biological mother of more than one son with hemophilia • A biological mother of one hemophilic son and has at least one other

blood relative with hemophilia• A sister of a male with hemophilia

46, XX 46, XY

Factor VIII allele - normal

Mutant VIII allele - normal

Germline/Gonadal Mosaicism

ovary testes

Factor VIIIFactor VIII

• Factor VIII geneFactor VIII gene– X-chromosome (Xq28), 186 kb, 26 exonsX-chromosome (Xq28), 186 kb, 26 exons

• 300 kDa protein:300 kDa protein:

• Biosynthesis: ?liver, ?lymphocytes, ?subset of ECsBiosynthesis: ?liver, ?lymphocytes, ?subset of ECs• Low concentration (100 ng/ml), bound to VWFLow concentration (100 ng/ml), bound to VWF

Molecular Defects in Hemophilia AMolecular Defects in Hemophilia A• >500 specific mutations identified:>500 specific mutations identified:

– http://europium.csc.mrc.ac.ukhttp://europium.csc.mrc.ac.uk– 1/3 new mutations (Haldane hypothesis)1/3 new mutations (Haldane hypothesis)– CpG dinucleotides = hot spot (~25% of point mutations)CpG dinucleotides = hot spot (~25% of point mutations)

• L1 insertion L1 insertion • Severe hemophilia A (FVIII<1%)Severe hemophilia A (FVIII<1%)

– gene deletions (5%)gene deletions (5%)– intron 22 inversion (45%) intron 22 inversion (45%) – point mutations (50%)point mutations (50%)

• ? Genetic modifiers -- VWF, FV Leiden ? Genetic modifiers -- VWF, FV Leiden

FVIII Gene Inversion (Intron 22)FVIII Gene Inversion (Intron 22)

• 45% of severe hemophilia A patients45% of severe hemophilia A patients

• Region particularly prone to rearrangement Region particularly prone to rearrangement – recurrent mutation eventrecurrent mutation event– recombination between repeated elements (gene A)recombination between repeated elements (gene A)

• Only occurs during male meiosisOnly occurs during male meiosis– Mother of “new” patient is generally a carrierMother of “new” patient is generally a carrier

Genetic Diagnosis for Hemophilia AGenetic Diagnosis for Hemophilia A• Prenatal diagnosisPrenatal diagnosis

– genetic consultationgenetic consultation– CVS, amniocentesis, cord blood samplingCVS, amniocentesis, cord blood sampling

• Screen for intron 22 inversionScreen for intron 22 inversion– only in severe patients (FVIII <1%)only in severe patients (FVIII <1%)

• Mutation screeningMutation screening– available through specialized DNA diagnostic labsavailable through specialized DNA diagnostic labs

• Linkage analysisLinkage analysis– informative in >90% of familiesinformative in >90% of families– requires other family members requires other family members – potential for incorrect diagnosis (recombination)potential for incorrect diagnosis (recombination)






platelet activation

crosslink formation

prothrombinase complex

VWF

VWF/Factor VIII InteractionVWF/Factor VIII Interaction

• VWF necessary for VWF necessary for FVIII stabilityFVIII stability

• Non-covalent complex Non-covalent complex • 1-2 FVIII per 100 1-2 FVIII per 100

VWF monomers VWF monomers • Plasma FVIII and VWF Plasma FVIII and VWF

levels proportional in levels proportional in normal and VWDnormal and VWD

Hoyer LW. NEJM, 330:38, 1994.

Type 2N VWD (VWD Normandy)

• Mutations in FVIII binding domain of VWFMutations in FVIII binding domain of VWF– decreased or absent FVIII binding activitydecreased or absent FVIII binding activity– normal adhesive functionnormal adhesive function

• Heterozygotes Heterozygotes – disproportionately low FVIIIdisproportionately low FVIII– co-inheritance with type 1 -- ? increased severityco-inheritance with type 1 -- ? increased severity

• HomozygotesHomozygotes– FVIII ~5-25% (? rare severe mutation)FVIII ~5-25% (? rare severe mutation)– mimics mild/ moderate hemophilia A, mimics mild/ moderate hemophilia A,

but autosomal recessivebut autosomal recessive– poor response to FVIII concentratespoor response to FVIII concentrates

• Test plasma VWF for FVIII bindingTest plasma VWF for FVIII binding• DNA testing available for limited set of DNA testing available for limited set of

type 2N VWF mutationstype 2N VWF mutations

Combined Deficiency of Factors V and VIIICombined Deficiency of Factors V and VIII• Rare autosomal recessiveRare autosomal recessive

– Most frequent in Jews of Sephardic and Middle Eastern originMost frequent in Jews of Sephardic and Middle Eastern origin

– > 100 families worldwide> 100 families worldwide

• Moderate bleeding tendencyModerate bleeding tendency– FV and FVIII antigen and activity ~5-30%FV and FVIII antigen and activity ~5-30%

• Mutations in ERGIC-53 (~75% of patients)Mutations in ERGIC-53 (~75% of patients)






Hemophilia BHemophilia B

• ~25% of hemophilia (incidence ~1:35,000 males)~25% of hemophilia (incidence ~1:35,000 males)• Phenotype indistinguishable from hemophilia APhenotype indistinguishable from hemophilia A• Severity related to factor IX levelSeverity related to factor IX level• Inhibitors correlate with type of mutationInhibitors correlate with type of mutation

– deletions > point mutationsdeletions > point mutations

• Hemophilia B LeydenHemophilia B Leyden– Severe hemophilia as childrenSevere hemophilia as children– Dramatic improvement at pubertyDramatic improvement at puberty

Factor IXFactor IX

• Factor IX geneFactor IX gene– X-chromosome (~ 10 cM from FVIII) 34 kb, 8 exonsX-chromosome (~ 10 cM from FVIII) 34 kb, 8 exons

• Serine protease (requires FVIII as cofactor: Xase complex)Serine protease (requires FVIII as cofactor: Xase complex)- Vitamin K-dependentVitamin K-dependent-carboxylated)-carboxylated)

• Biosynthesis: liverBiosynthesis: liver• Plasma concentration ~10 Plasma concentration ~10 g/mlg/ml

Hemophilia B: GeneticsHemophilia B: Genetics• X-linked inheritanceX-linked inheritance

– ~1/3 patients represent new mutations (Haldane hypothesis)~1/3 patients represent new mutations (Haldane hypothesis)

• Germinal mosaicismGerminal mosaicism

• Low FIX in female consider:Low FIX in female consider:– skewed X-inactivationskewed X-inactivation

– chromosomal abnormality (normal X inactivated)chromosomal abnormality (normal X inactivated)

Molecular Defects in Hemophilia BMolecular Defects in Hemophilia B• >680 specific mutations identified:>680 specific mutations identified:

– http://www.umds.ac.uk/molgen/http://www.umds.ac.uk/molgen/– 1/3 new mutations (Haldane hypothesis)1/3 new mutations (Haldane hypothesis)– CpG dinucleotides=hot spot (~1/3 of point mutations)CpG dinucleotides=hot spot (~1/3 of point mutations)– 425 different amino acid substitutions425 different amino acid substitutions– Mutations at 9/12 Gla codonsMutations at 9/12 Gla codons

• Large deletions: increased risk of inhibitor developmentLarge deletions: increased risk of inhibitor development• Estimate of human mutation frequency:Estimate of human mutation frequency:

– 2.14 X 102.14 X 10-8 -8 per base per generationper base per generation– 128 mutations/zygotes (1% detrimental)128 mutations/zygotes (1% detrimental)

• Mutation screeningMutation screening– available through specialized DNA diagnostic labsavailable through specialized DNA diagnostic labs

• Linkage analysisLinkage analysis

History of Treatment for Hemophilia

1950s A basic understanding of coagulation1960s Cryoprecipitate1970s Freeze-dried concentrates from pooled plasma

Increase in viral inactivation efforts1980s More advanced viral inactivation procedures

Expanded donor screening/testing Increased concentration/purity

1990s Non–plasma Recombinant DNA concentratesMore sensitive viral marker screening tests

2000s Gene therapy?

Dr. Graham Pool discovered factor

VIII-rich cryoprecipitate

Goals of Hemophilia Care

• Prevention: Education, prophylaxis, and/or physical fitness with injury avoidance.

• Prompt self-treatment: Patients become quite adept at prompt preventive and emergency self-treatment.

• Rehabilitation: To limit secondary musculoskeletal and neurologic complications once the bleed subsides.

C. Harris, age 14

Treatment Basics

• Infuse concentrated Factor VIII• 80% of patients do at home • dose based on weight (eg. 2% rise /unit /kg) - • don’t need levels of 100%.

• DDAVP (IV/nasal) (mild hemophilia A) • releases factor VIII from endothelial cells • doubles or triples plasma factor VIII level

D. Musher, age 10

Recombinant FactorVIII:

Insertion of human factor VIII DNA into vector system

allowing incorporation into non-human mammalian cell

lines for continued propagation

HEMOPHILIA THERAPY IMPROVEMENTS:Volumes (mL) required to obtain a factor VIII dose of 2000 IU:

Whole Blood >4000 mlWhole Plasma = 2000 mlCryoprecipitate = 400 mlEarly Concentrates = 80 mlToday’s Concentrates <20 ml

Jason Burdick, age 12, Green Bay, WI

Financial & Insurance Issues

• > 70% of clotting factor distribution is by for-profit companies average cost/yr for human plasma derived or recombinant factor is $50,000 - $100,000

• Prophylaxis requires about 150,000 units/yr for a 65-pound child costing $85,000 per year

• Prophylaxis is covered by insurance on a case-by-case basis.

Avoid drugs that aggravate bleeding problems: • Aspirin • Heparin • Warfarin • Nonsteroidal anti-inflammatory drugs

Hemophilia is an ideal disease for gene therapy:

• caused by a single malfunctioning gene • just small increase in factor level will provide great benefit:

raising factor by 2% will prevent spontaneous hemorrhages into joints, brain and other organs; levels greater than 20% to 30% will prevent bleeding in most injuries

Molecular Genetics of HemophiliaMolecular Genetics of HemophiliaSummarySummary

• Hemophilia A: mutations in FVIII geneHemophilia A: mutations in FVIII gene– Intron 22 inversion (~45% of severe)Intron 22 inversion (~45% of severe)

– Other deletions and point mutationsOther deletions and point mutations

• Other Genetic Disorders with low Factor VIIIOther Genetic Disorders with low Factor VIII– Type 2N VWD: mutations in VWD factor VIII binding domainType 2N VWD: mutations in VWD factor VIII binding domain

– Combined Deficiency of Factor V and Factor VIIICombined Deficiency of Factor V and Factor VIII• ERGIC-53 gene mutations, ?other geneERGIC-53 gene mutations, ?other gene

• Hemophilia B: mutations in Factor IXHemophilia B: mutations in Factor IX

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