HEMOPHILIA (The Royal Disease)MLT 429

Acknowledgement

With the name of ALLAH, the most Gracious and Merciful

My most gratitude goes to ALLAH as I have successfully completed the task given for

this subject, MLT 429; Fundamentals of Genetics and Cellular Biology-Hemophilia (The Royal

Disease) on the time that specified. First of all, an infinity of thanks to my beloved mother, Pn.

Siti Jamaliah bt. Musa for being my life inspiration and giving her maximum support; morals and

materials in my study. Not to be forgotten, I would like also thank to our dedicated lecturer of

this subject, En. Mohd Fahmi b. Mastuki for giving us full guidelines in completing the task and

being very considerable. Last but not least, thanks to all of my colleagues who have direct or

indirectly contributed in accomplishing the assignment.

Nor Azean bt. Abdul Latif

(2008749073)

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HEMOPHILIA (The Royal Disease)MLT 429

INTRODUCTION

Queen Victoria met Prince Albert of Saxe-Coburg when they were both seventeen. She

fell deeply in love with him and several years later, Victoria proposed to Albert. On February 11,

1840 they were married. Victoria made Albert Prince Consort, giving him the equal say to rule

the country and live deliriously happy. However, everything was changed as she delivered her

eighth child, Leopold. He was born with a very non-familiar disease at that time. Prince Leopold

was easily bruises and it took such a very long time to heal. The most severe part, he always

had prolonged bleeding. Victoria strongly believed that the disease is not in her family as none

of her family has a history of such disease. She became overprotective of her son, coddling him

and hovering over him as many parents of ill children do. However, he died of the disease at the

age of 31 after falling and had a brain hemorrhage. The ‘curse’ did not stop there as the disease

was passed to several generations of her heirs. The disease even cause to Bolshevik

Revolution, changing the course of history and destroying the lives of millions of people. The

disease was popularly called ‘The Royal Disease’ during Victorian and now it is scientifically

known as Hemophilia.

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HEMOPHILIA (The Royal Disease)MLT 429

OVERVIEW

Hemophilia is a type of hereditary genetic deficiencies. It impairs the ability of the body

to control blood clotting or coagulation as a mechanism to stop bleeding when a blood vessel is

broken. There are two types of Hemophilia and they are categorized according to the clotting

factor that they deficit of. The most common form with the prevalence of 1 in 5,000-10,000 male

births is Hemophilia A which factor clotting VIII is absent. Another form is Hemophilia B (also

known as Christmas disease) which caused by Factor IX deficiency and it occurs in 1 in about

20,000–34,000.

Pathogenesis

These genetic deficiencies may lower blood plasma clotting factor levels of coagulation

factors needed for a normal clotting process. When a blood vessel is broken, platelets stick

together to form a plug (fibrin formation), but the missing coagulation factors (Factor VIII or IX)

prevent fibrin formation which is necessary to maintain the blood clot. Thus a hemophiliac does

not bleed more intensely than a normal person, but for a much longer amount of time. In severe

hemophiliacs even a minor injury could result in blood loss lasting days, weeks, or not ever

healing completely. The critical risk here is with normally small injuries which, due to missing

Factor VIII or IX, take long times to heal. In areas such as the brain or inside joints this can be

fatal or permanently debilitating.

Fibrin clot formation

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HEMOPHILIA (The Royal Disease)MLT 429

Etiology

The genes of the disease are carried on X chromosome (X-linked), therefore there are

different patterns of expression in females and males, because a female has two X

chromosomes which inherited one from her father and another from her mother. A male just get

one X chromosome which inherited from his mother as from his father, he inherited the Y

chromosome. They are referred as hemizygous1 for X linked traits. In females, X-linked traits

are passed just like autosomal traits whereby two copies are needed for expression of a

recessive allele. However, in males, a single copy of an X-linked allele causes expression of the

trait or disease because there is no copy of the gene as a ‘back-up’ on a second X chromosome

to mask the other’s effect. Therefore, females are almost exclusively carriers of the disorder,

and may have inherited it from either their mother or father. A woman only can be affected if she

received the affected X chromosome from her hemophiliac father and a carrier mother.

Hereditary X-linked genetic disorder

1 Describing genes that are carried on an unpaired chromosome (sex chromosome) for example the genes on the X chromosome in males

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HEMOPHILIA (The Royal Disease)MLT 429

Hemophilia A

The defective gene in hemophilia A is F8 which is located near the tip of the long arm of

the X chromosome (Xq28). The gene consists of 26 exons and 25 introns. These encode an

8.8kb of mRNA. The majority of exons are small, being at most a few hundred nucleotides in

length. Exons 14 and 26 are rather larger, particularly exon 14, which is 3kb in length. Exon 14

encodes the B domain of the Factor VIII protein. Introns 1 and 22 are notable for their large size

and presence of repeated sequences, of which copies are present elsewhere on the X

chromosome. These repeated regions are involved in the two intrachromosomal inversion

mutations:

F8 intron 22 inversion

F8 intron 1 inversion

The Factor VIII protein has a repeated domain structure, with triplicated homology region A (A 1,

A2, A3), a (acidic) and C domains (duplicated homology region; C1, C2), plus a single large

glycosylated B domain which is removed when Factor VIII.

Factor VIII gene and protein

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HEMOPHILIA (The Royal Disease)MLT 429

F8 intron 22 inversion (flip-tip inversion)

The F8 gene is located at Xq28, towards the telomere. Based on the figure above, the

F8 gene is shown in grey. There are three copies of gene A in this region where one within in

intron 22 and two near the telomere. These regions are referred to as intron 22 homologous

regions (int-22h) and are numbered 1-3.During spermatogenesis at meiosis, the single X pairs

with the Y chromosome in the homologous regions. The X chromosome is longer than the Y

and there is nothing to pair with the most of the long arm X. The chromosome undergoes

homologous recombination where the 5’ 22 exons of F8 have been relocated to the telomeric

end of the X chromosome, whilst the 3’ four exons remain in their original location. The F8 gene

is thus split into two parts, facing in opposite orientations, and separated. This split F8 gene

cannot encode functional Factor VIII protein and result to severe Hemophilia A.

Summary of F8 intron inversion (flip-tip inversion)

Result from homologous intrachromosomal recombination

Inversion mutation occurs de novo2 once per 10,000 male meiosis

Every ejaculate contains at least one sperm with an F8 intron 22 inversion mutation

Responsible for 45% of severe Hemophilia A

F8 intron 1 inversion

2 Spontaneous mutation which is not caused by a mutagen and usually originates as an error in DNA replication. Queen Victoria appears to have been a de novo mutation and is considered the source of this line of the disease

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HEMOPHILIA (The Royal Disease)MLT 429

As explained earlier, the F8 gene is located at Xq28, towards the telomere (tel) of the

long arm of the X chromosome. Intron 1 is repeated once, 5’ and telomeric to F8. The looping

round of the X chromosome, is probably occurs particularly at male meiosis, similar to the intron

22 inversion. Two copies of int-1h line up and homologous recombination can occur between

them, as the sequences are >99% identical. Recombination is observed much less frequently

with the intron 22 inversion. This is likely due to physical constraints for intron 1 inversion. As

the result of the inversion is the promoter and exon 1 of F8 have been relocated to the telomeric

end of the X chromosome, whilst the remainder of the F8 gene stays in its original location. The

F8 gene is thus split into two parts, facing in opposite orientations. This split F8 gene cannot

encode functional Factor VIII protein and result to severe Hemophilia A.

Summary of F8 intron 1 inversion

Similar to intron 22 inversion

Results in F8 gene lacking a promoter and first exon

Responsible for approximately 2% of severe Hemophilia A

Intrachromosomal inversions make up of 50% causes of severe Hemophilia A cases. Both

inversions are seen at approximately the same frequency in all populations worldwide. The

intron 22 inversion is far more prevalent than the intron 1 inversion.

Other mutation type that lead to Hemophilia A

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HEMOPHILIA (The Royal Disease)MLT 429

Point mutation

The majority of non-inversion cases of Hemophilia A, and nearly all cases of Hemophilia

B result from point mutations, affecting a single nucleotide. Further explanation will be described

later under the topic of Hemophilia B.

Deletion of part or the entire gene

Up to 5% of patients with severe hemophilia have a deletion of all or part of the F8 or F9

gene. However, deletions of one or a few exons are more common than whole gene deletions.

No normal Factor VIII or IX can be produced in these individuals, thus result to severe disease.

Insertion into gene (repetitive sequence)

A very small number of patients have an insertion of a repetitive element (LINE1 or Alu)

into the gene. This causes severe hemophilia by disrupting the coding sequence of the gene.

Splicing error effecting production of mRNA

Point mutations can affect splicing of pre-mRNA to mature mRNA. There are two

virtually invariant nucleotides at each end of each intron; GT at the 5’ end (splice donor) and AG

at the 3’ end (splice acceptor). Alterations of these nucleotides will almost always disrupt

splicing and cause severe (sometimes moderate) disease. Alteration of other nucleotides near

the splice site can result in less severe disease. Exonic splice enhancer sites can also be

altered by point mutation; some apparently silent (no amino acid substitution) mutations, for

example in exon 11 of the F8 gene, have been shown to disrupt splicing. Potential splice

mutations can be predicted using web-based splice prediction software.

Hemophilia B

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HEMOPHILIA (The Royal Disease)MLT 429

The F9 gene is considerably smaller than the F8 gene. Its 8 exons encode a serine

protease enzyme, responsible for cleavage of Factor X to Factor Xa.

Factor IX gene and protein

Point mutation

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HEMOPHILIA (The Royal Disease)MLT 429

A G>A nucleotide substitution predicts replacement of Cysteine (Cys) by Tyrosine (Tyr).

Most hemophilia patients, with both hemophilia A and B, have a missense mutation. These

alterations can be anywhere in the F8 or F9 genes. Disease severity depends on the location

and any particular function of the amino acid affected. A C>T substitution, predicts replacement

of Arginine (Arg) by a premature termination codon (stop or X). Nonsense mutations prevent

any Factor VIII or Factor IX being made, so always result in severe hemophilia. The loss of an A

nucleotide from a run of A nucleotides by which such repetitive sequences are always prone to

DNA replication errors, with a nucleotide more frequently being lost than gained. F8 particularly

has several runs of the A nucleotides. Recurrent deletions (and insertions) are seen in each of

these. They result in severe (and occasionally moderate) disease in each of these. They result

in severe (and occasionally moderate) disease.

Hemophilia B Leiden

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HEMOPHILIA (The Royal Disease)MLT 429

Transcription factor binding sites in the F9 gene promoter

Hemophilia B Leiden mutations affect the binding of transcription factors C/EBP or HNF4

to the promoter region. Individuals with nucleotide substitutions between -20 and +13, relative to

the transcription start site, have been reported with this phenotype of hemophilia B. Following

puberty, binding to the androgen response element starts to ameliorate3 hemophilia, as some

F9 transcription becomes possible. Individuals with a point mutation at -26 (Brandenburg

mutation) have lifelong hemophilia B. Before puberty, the mutation blocks binding to the HNF4

site.

Following puberty, it blocks binding to the androgen response element.

Summary of Hemophilia B Leiden

An unusual form of Hemophilia B which seen in 3% of Hemophilia B patients.

The patients of Hemophilia B Leiden have Factor IX level which increase at puberty (age-

dependent rise)

From puberty, the Factor IX level mirroring that in normal males is seen.

The baseline Factor IX level and the extent of the rise in level is dependent upon the F9

mutation

DIAGNOSIS

3 To improve or recover something

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HEMOPHILIA (The Royal Disease)MLT 429

The diagnosis of Hemophilia A and B cannot be made on clinical findings. A coagulation

disorder is suspected in individuals with any of the following:

Hemarthrosis, especially with mild or no antecedent trauma

Deep-muscle hematomas

Intracranial bleeding in the absence of major trauma

Neonatal cephalohematoma or intracranial bleeding

Prolonged oozing or renewed bleeding after initial bleeding stops following tooth

extractions, mouth injury, or circumcision *

Prolonged bleeding or renewed bleeding following surgery or trauma *

Unexplained GI bleeding or hematuria *

Menorrhagia, especially at menarche *

Prolonged nosebleeds, especially recurrent and bilateral *

Excessive bruising, especially with firm, subcutaneous hematomas

* Any severity; otherwise, especially in more severely affected persons

SYMPTOMS

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HEMOPHILIA (The Royal Disease)MLT 429

Severity Factor VIII

Clotting Activity Symptoms

Severe <1% Frequent spontaneous bleeding; abnormal bleeding after

minor injuries, surgery, or tooth extractions

Moderatel

y

severe

1%-5% Spontaneous bleeding is rare; abnormal bleeding after minor

injuries, surgery, or tooth extractions

Mild >5%-35% No spontaneous bleeding; abnormal bleeding after major

injuries, surgery, or tooth extractions

Symptoms Related to Severity of Hemophilia A

SeverityFactor IX Clotting

Activity Symptoms

Severe <1% Frequent spontaneous bleeding; excessive and/or prolonged

bleeding after minor injuries, surgery, or tooth extractions

Moderatel

y severe 1%-5%

Spontaneous bleeding rare; excessive and/or prolonged

bleeding after minor injuries, surgery, or tooth extractions

Mild >5%-30% No spontaneous bleeding; excessive and/or prolonged

bleeding after major injuries, surgery, or tooth extractions

Symptoms Related to Severity of Hemophilia B

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HEMOPHILIA (The Royal Disease)MLT 429

PREVENTION

Prevention of primary manifestations

Children with severe Hemophilia A and B are often each given "primary" prophylactic

infusions of Factor VIII or IX concentrate two to three times a week to maintain Factor VIII and

IX clotting activity above 1%; these infusions prevent spontaneous bleeding and decrease the

number of bleeding episodes. Prophylactic infusions almost completely eliminate spontaneous

joint bleeding, decreasing chronic joint disease, although complications of venous access ports

in young children can occur.

Prevention of secondary complications

Prevention of chronic joint disease is a major concern. Controversy still exists as to

whether all individuals with severe Hemophilia A and B benefit from primary prophylaxis and,

especially, whether the benefits of primary prophylaxis justify the risk of an indwelling venous

catheter in a young individual. "Secondary" prophylaxis is often used for several weeks, even in

adults, if recurrent bleeding in a "target" joint or synovitis occurs.

Both Hemophilia A and B patients also must avoid the activities that involve a high risk of

trauma, particularly head injury and consumption of aspirin or aspirin-containing products.

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HEMOPHILIA (The Royal Disease)MLT 429

SCREENING

Both Hemophilia A and B can be screened by Coagulation Screening Test. Evaluation of an

individual with a suspected bleeding disorder comprised of:

Platelet count and bleeding time or platelet function analysis (PFA closure times)

Activated Partial Thromboplastin Time (APTT)

Thrombin Time (PT)

Thrombin Time (TT) and/or plasma concentration of fibrinogen (useful for rare disorders).

In individuals with Hemophilia A and B, the above screening tests are normal, with the

exceptions that the APTT is prolonged in severe and moderately Hemophilia A and B. But, in

mild form of both Hemophilia types the APTT may be normal. However, in many clinical

laboratories, the APTT is not sensitive enough to diagnose mild form of bleeding disorders.

Confirmation Diagnosis:

Specific Coagulation Factor Assay

Normal Hemophilia A Hemophilia B

Factor VIII clotting

activity

50%-150% <30%-35% <30%

In vitro Factor VIII

clotting activity

(Classification)

_ Severe:<1% Factor

VIII

Severe:<1% Factor IX

Moderate:1%-5%

Factor VIII

Moderate:1%-5%

Factor IX

Mild: 6%-35% Factor

VIII

Mild:>5%-30% Factor

IX

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HEMOPHILIA (The Royal Disease)MLT 429

Molecular Genetic Testing

Hemophilia A

Test Method Mutations Detected

Targeted mutation analysis

F8 intron 22 inversion

F8 intron 1 inversion

Mutation scanning or sequence analysis F8 sequence variants

Deletion analysis F8 exonic and large gene deletions

Hemophilia B

Test Method Mutations Detected

Sequence analysis F9 sequence variants

Deletion/ duplication analysis F9 exonic and large gene deletions

Molecular Genetic Testing is also available for the carrier screening of most at-risk females if

the mutation has been identified in the family.

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HEMOPHILIA (The Royal Disease)MLT 429

Prenatal Testing

Prenatal testing is available for pregnancies of women who are carriers if the mutation

has been identified in a family member or if linkage has been established in the family. The

usual procedure is to determine fetal sex by performing chromosome analysis of fetal cells

obtained by chorionic villus sampling (CVS) at approximately ten to 12 weeks' gestation or by

amniocentesis usually performed at approximately 15-18 weeks' gestation. If the karyotype is

46,XY, DNA extracted from fetal cells can be analyzed for the known F8 or F9 disease-causing

mutation or for the informative markers.

Percutaneous Umbilical Blood Sampling (PUBS).

If the disease-causing F8 or F9 mutation is not known and if linkage is not informative,

prenatal diagnosis is possible using a fetal blood sample obtained by PUBS at approximately

18-21 weeks' gestation for assay of Factor VIII clotting activity. However, the requests for

prenatal testing for conditions such as Hemophilia A or B that do not affect intellect and have

treatment available are not common. Differences in perspective may exist among medical

professionals and within families regarding the use of prenatal testing, particularly if the testing

is being considered for the purpose of pregnancy termination rather than early diagnosis.

Preimplantation Genetic Diagnosis (PGD)

This test may be available for families in which the disease-causing mutation has been

identified in an affected family member.

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HEMOPHILIA (The Royal Disease)MLT 429

Linkage analysis

Tracking an unidentified F8 or F9 mutation.

When a disease-causing mutation of the F8 or F9 gene is not identified in an affected

family member by direct DNA testing, linkage analysis can be considered to obtain

information for genetic counseling in families in which more than one family member has the

unequivocal diagnosis of Hemophilia A or B. Linkage studies are always based on accurate

clinical diagnosis of Hemophilia A or B in the affected family members and accurate

understanding of the genetic relationships in the family. In addition, linkage analysis depends

on the availability and willingness of family members to be tested and on the presence of

informative heterozygous polymorphic markers. Use of up to five intragenic polymorphisms

and one extragenic polymorphism is informative in approximately 80%-90% of families.

Recombination events between F8 and the extragenic site occur in up to 5% of meioses, but

have not been observed between hemophilic mutations and intragenic sites.

Identifying the origin of a de novo mutation.

Among the nearly 50% of families with a simplex case of Hemophilia A and B (i.e.,

occurrence in one family member only), the origin of a de novo mutation can often be

identified by performing molecular genetic testing in conjunction with linkage analysis. The

presence of the mutation on the affected individual's Factor VIII or IX haplotype is tracked

back through the parents and, if necessary, through maternal grandparents to identify the

individual in whom the mutation originated.

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HEMOPHILIA (The Royal Disease)MLT 429

CONCLUSION

As Hemophilia is an inherited genetic disorder, it cannot be prevented. It is best for the

hemophiliacs to seek for medical geneticist and genetic counselor for health consultation. The

Hemophiliacs must maintain a healthy body weight to minimize the pressure on the joints which

may lead to bleeding episodes. A safe exercise plan must be created with the doctor to

strengthen the joints and muscles which can help to prevent bleeding. It is important to prevent

bleeding into joints as it may result in severe disability. Marrying a Hemophilia patient or a

carrier is not a prohibition. However, the couples must have a thorough knowledge in family

planning to avoid continuous inheritance of the affected genes to their offspring. Besides of

choosing to have none, now a free- hemophilia affected gene offspring can be born as the

technology of genetic has been more enhanced nowadays. The advent of Preimplantation

Genetic Diagnosis (PGD) allows Hemophilic or carrier parents choose which embryos to implant

in the mother’s womb based on genetic test results, thus stop the inheritance of the affected

gene. The invention of gene therapy to alter the genes of an inherited disorder also gives a

longer-lasting effect than treating the symptoms and there’s hope to the Hemophiliacs to stay

more healthily, safely and less-risky.

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HEMOPHILIA (The Royal Disease)MLT 429

References:

1. Lewis R. (2007) Human Genetics Concept and Applications, Seventh Edition, McGraw

Hill U.S.A

2. A.V. Hoffbrand, J.E. Petit, & P.A.H Moss (2001) Essential Hematology, Fourth Edition,

Blackwell Publishing U.K

3. E. A. Martin (2002) Oxford Concise Medical Dictionary, Sixth Edition, Oxford University

Press N.Y

4. http://en.wikipedia.org/wiki/Haemophilia retrieved: August 23rd, 2009, 6:15 AM

5. http://en.wikipedia.org/wiki/Haemophilia_in_European_royalty retrieved: September 3rd,

2009, 10:57 PM

6. http://ghr.nlm.nih.gov/condition=hemophilia retrieved: September 4th, 2009, 1:42 AM

7. http://www.genome.gov/20019697 retrieved: August 24th, 2009, 1:52 AM

8. http://en.wikipedia.org/wiki/Preimplantation_genetic_diagnosis retrieved: September 13th,

2009, 2:19 AM

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