Hemophilia In Genetic Preview

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 BiologyHemophilia (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 Etiology 3

HEMOPHILIA (The Royal Disease) MLT 429 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

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 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 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 (A1, 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

F8 intron 22 inversion (flip-tip inversion)

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HEMOPHILIA (The Royal Disease) MLT 429

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

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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 F8 intron 1 inversion

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. 7

HEMOPHILIA (The Royal Disease) MLT 429

Other mutation type that lead to Hemophilia A ✔ 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

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HEMOPHILIA (The Royal Disease) MLT 429 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 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.

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HEMOPHILIA (The Royal Disease) MLT 429

Hemophilia B Leiden

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

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To improve or recover something

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HEMOPHILIA (The Royal Disease) MLT 429

DIAGNOSIS 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 12

HEMOPHILIA (The Royal Disease) MLT 429

Factor VIII Severity

Clotting

Symptoms

Activity

Severe

<1%

Moderate ly

1%-5%

severe

Mild

>5%-35%

Frequent spontaneous bleeding; abnormal bleeding after minor injuries, surgery, or tooth extractions

Spontaneous bleeding is rare; abnormal bleeding after minor injuries, surgery, or tooth extractions

No spontaneous bleeding; abnormal bleeding after major injuries, surgery, or tooth extractions

Symptoms Related to Severity of Hemophilia A

Factor IX Severity

Clotting

Symptoms

Activity

Frequent spontaneous bleeding; excessive and/or Severe

<1%

prolonged bleeding after minor injuries, surgery, or tooth extractions

Moderate ly severe

Mild

Spontaneous bleeding rare; excessive and/or 1%-5%

prolonged bleeding after minor injuries, surgery, or tooth extractions

>5%-30%

No spontaneous bleeding; excessive and/or 13

HEMOPHILIA (The Royal Disease) MLT 429

prolonged bleeding after major injuries, surgery, or tooth extractions Symptoms Related to Severity of Hemophilia B

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 aspirincontaining 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

Factor VIII

Normal

Hemophilia A

Hemophilia B

50%-150%

<30%-35%

<30%

_

Severe:<1% Factor

Severe:<1% Factor

VIII

IX

Moderate:1%-5%

Moderate:1%-5%

Factor VIII

Factor IX

clotting activity In vitro Factor VIII clotting activity (Classification)

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HEMOPHILIA (The Royal Disease) MLT 429 Mild: 6%-35%

Mild:>5%-30%

Factor VIII

Factor IX

✔ Molecular Genetic Testing Hemophilia A

Test Method

Mutations Detected

F8 intron 22 inversion Targeted mutation analysis F8 intron 1 inversion

Mutation scanning or sequence analysis

Deletion analysis

F8 sequence variants

F8 exonic and large gene deletions

Hemophilia B

Test Method

Mutations Detected

Sequence analysis

F9 sequence variants

Deletion/ duplication

F9 exonic and large gene

analysis

deletions

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HEMOPHILIA (The Royal Disease) MLT 429 Molecular Genetic Testing is also available for the carrier screening of most at-risk females if the mutation has been identified in the family.

✔ 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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