Hemophilia A

BTEC 3301 Principles of Genomics Proteomics and Bioinformatics

Adiaratou Sidibe - adiarsi20@yahoo.fr - 0835640
Shane Poole Shanepoole50@gmail.com - 1041878
Introduction
Hemophilia A is a known X-linked recessive disorder. This
condition or bleeding disorder is characterized by a deficiency in the
activity of a coagulation factor, which in this case is F8 or coagulation
factor VIII. This condition is clinically known to be heterogeneous and
its severity depends on the plasma level of the coagulation factor VIII.
Varying levels of hemophilia exist which are categorized based on
percentage of coagulation factor within blood plasma compared to
normal levels.
According to the CDC website, Hemophilia, is a hereditary
bleeding disorder which affects mostly males due to X chromosome
singularity. Hemophilia is a medical condition in which the blood
clotting becomes a challenge for those affected. Symptoms include
spontaneous bleeding from injuries or surgery. Blood contains many
proteins called clotting factors that assist in blood coagulation,
however; those individuals with hemophilia have low levels of either
factor VIII or factor IX. The severity of the condition is determined by
the amount of factor in the blood thus the lower the amount of the
factor, the higher the chances that spontaneous bleeding will occur
leading to more severe health complications.
If this disease causes such problems, one may wonder what
causes it? Many fanatics of life science have established that
hemophilia is the consequence of a mutation or change in one of the
genes. This gene is the gene that provides instructions for making the
clotting factor proteins needed to form a blood clot. As a result of this
change or mutation, the clotting protein may not work properly as it
should or maybe lacking function altogether. These genes, which are
so significant for the clotting proteins function, are located on the X
chromosome. Genetically males have one X and one Y chromosome
while their opposites in sex, females, have two X chromosomes. Males
tend to display the phenotype more because females with only one
mutated X chromosome dont display the phenotype but are carriers.
Hemophilia A can be suspected by prolonged bleeding after tooth
extraction, or surgery, an overflow of blood due to injuries, and
postponed or recurrent bleeding before complete wound healing. In

severe hemophilia A, characterized by spontaneous joint or deepmuscle bleeding, patients must be diagnosed during the first two years
of life; without prophylactic treatment they should expect to
experience an average of two to five spontaneous bleeding episodes
each month while those with moderate hemophilia A almost never
have spontaneous bleeding; however, they do not experience
lengthened or delayed bleeding after relatively minor trauma and are
often diagnosed before age five to six years; the frequency of bleeding
episodes varies, usually from once a month to once a year, on the
other hand individuals with mild hemophilia seldom experience
spontaneous bleeding episodes; however, without pre- and
postoperative treatment, abnormal bleeding occurs with surgery or
tooth extractions; the frequency of bleeding episodes varies widely,
typically from once a year to once every ten years and are not
diagnosed until later in the life.
When faced with these signs and symptoms please contact a
health professional immediately. The various and multiple symptoms of
hemophilia include bleeding into the joints, which may result in
swelling and pain or tightness in the joints. Often, swelling occurs in
the knees, elbows, and ankles. Another symptom could be bleeding
into the skin, muscle, or soft tissue causing building-up of blood in that
area, this condition is referred to as hematoma. Sometimes, bleeding
could occur in the mouth and gums and after tooth extraction bleeding
can be hard to stop. Circumcision becomes fetal or dangerous when
affected. Other miscellaneous situations would be bleeding after
vaccination, bleeding in infants head after a difficult delivery, frequent
appearance of blood in bowel movement and urine, and arduous
nosebleed. Hemophilia Bleeding can be within joints, which could
engender a chronic joint disease; bleeding in the brain can trigger
other adverse long-term effects. Those conditions include seizures,
paralysis, and even death if the bleeding is not stopped or if bleeding
takes place in a vital organ of the body.
It was established that individuals with hemophilia A experience
more recurrent bleeding episodes in childhood and adolescence than in
adulthood. Genetic studies have shown that close to 10% of carrier
females are at risk for bleeding even when those related family
members were mildly affected. For that reason, symptomatic carriers,
although symptoms are usually mild can show after major trauma or
invasive procedures a prolonged or excessive bleeding behindhand of
severity.

The diagnosis of hemophilia A is accomplished in individuals with

low factor VIII clotting activity in the presence of a normal factor, which
is known as Willebrand factor (VWF) level. The diagnosis of hemophilia
A is established by a genetic testing referred to as molecular genetic
testing, which tests for the clotting factor F8, the gene encoding factor
VIII, and showed pathogenic variants in as many as 98% of individuals
with hemophilia A. Other testings include: Coagulation screening tests,
Coagulation factor assays, Complete Blood Count, Activated Partial
Thromboplastin Time Test, Prothrombin Time Test, Fibrinogen Test, and
Clotting Factor Tests.
Based on information available on gene view and CDC homepage, on
the most effective way to treat hemophilia is to replace the missing
blood clotting factor to allow blood clotting appropriately. This is done
by injecting commercially prepared clotting factor concentrates into a
patients vein. There are two major types of merchandised clotting
factor, the first being intravenous infusion of factor VIII concentrate
and the second being recombinant factor VIII concentrates. The way
intravenous infusion of factor VIII concentrate works is that all blood
components and parts such as plasma are consistently tested for the
viruses. When no viruses present, the clotting proteins are isolated
from other parts of the plasma, purified processed into a freeze-dried
product. This freeze-dried product is analyzed and treated to kill any
potential pathogens or viruses prior to usage.
Methods
To better understand how Hemophilia A is inherited we need to
identify what mutations are pathogenic. The first step is to find the
normal genomic sequence for the encoded protein. Using NCBI we first
searched the OMIM database for Hemophilia A. From this page we were
able to easily identify the F8 gene and associated coagulation factor
VIII protein. The NCBI F8 gene page designates the normal mRNA
transcript and functional encoded protein, coagulation factor VIII
isoform a precursor. The associated NCBI protein page leads to the
UniProt FA8_Human page that contains several important information
sections. The function of the protein is summarized as well as the
molecular and biological functions. The normal amino acid sequence
for both isoforms are available, as well as key protein domains and
associated InterPro domain analysis pages. We analyzed the protein
sequence with a protein BLAST to identify protein homologs. A DELTA-

Blast was used as well to identify sequences less homologous to the

original. We can apply statistical reasoning to the BLAST results to
identify homologous proteins within other species. We also utilized a
dot plot analysis to compare the normal protein sequences to the
mutant one. UniProt contains a section that details amino acid
substitution variants that result in Hemophilia A. There were many
changes that result in pathogenic hereditary factor VIII deficiency
disease. Missense and frameshift mutations are the largest categories
of changes that result in improper coagulation factor VIII protein
function. From this list we selected a highly reviewed missense variant
to further study what changed within the protein and mRNA sequence
to make it non-functional and cause hemophilia.
Results
Hemophilia A is caused by a multitude of genetic changes within
the source gene. The human coagulation factor VIII (F8) gene is our
gene of interest. Starting at the NCBI gene result page for the F8
coagulation factor VIII, the gene is located on chromosome X within the
q28 region. The normal mRNA encodes a protein, coagulation factor
VIII isoform A. The mrna sequence is 9048 base pairs long and contains
27 exons with coding region ranging from base 172 to 7227. Analyzing
the uniprot page for the protein we can gather a better grasp of the
protein function. Coagulation factor VIII is a vital coagulation cofactor
as it is essential in activation of coagulation factor ixa. The protein
contains 2351 amino acids and 11 domains. Processing can be done to
the transcript to generate 2 heavy chain isoforms, a B chain, and light
chain isoform. Molecular functions of this protein include copper ion
binding, oxidoreductase activity, and serine endopeptidase action.
These functions allow it to participate in blood coagulation, cell
adhesion, and platelet activation activities. Several domains within the
protein allow it to have these functions. The Plastocyanin-like domain
allows the protein to have copper ion binding and electron carrier
activity. This domain enables the factor to bind a copper ion cofactor
important for associated redox reactions (4). The F5/8 C-terminal type
domain within coagulation factor VIII contains about 150 amino acids
and assists with cell adhesion. Specifically this domain allows the
protein to bind anionic phospholipids on the surface of platelets and
endothelial cells (5). Uniprot identifies these domains as well as lists
their specific positions within the proteins amino acid sequence. We
chose to review a highly studied missense mutation within the

sequence that altered a codon. Within the mRNA sequence the

mutation occurred at position 1343, a guanine was replaced with an
adenine. This resulted in a new codon that replaced the wild type
arginine residue with a histidine residue at amino acid position 391
within the protein. The 391 - 392 amino acid site is important to this
protein as it acts as a thrombin cleavage site. Thrombin is a protease
that cleaves bonds after arginine or lysine and is responsible for
activating blood coagulation factors (11). Since the wild type arginine
has been substituted this cleavage site is inactivated and factor VIII
loses procoagulant function resulting in Hemophilia A. The dot plot
(figure section) is difficult to see in the context of this mutation
because it is a single amino acid change in protein sequence. The
dotplot contains a horizontal line indicating high sequence similarity.
The lines on the side represent sequence similarity within other parts
of itself. The BLAST results (figures section) returned homologous
coagulation factors in other species. Even proteins that are not similar
share domains that function analogously. This 391 mutation has
become an important disease marker for Hemophilia, as several
methods have been developed to directly characterize it specifically.
An immunoadsorbent method was researched that can analyze blood
plasma and detect variant coagulation factor VII based on heavy chain
accumulation when incubated with thrombin (13).
Discussion:
There is currently research aimed to study the hereditary disease
known as hemophilia A. In the course of this research the gene for the
disease, the protein, and the mRNA were evaluated to draw a
conclusion on the different variation that took place in the normal
genetic code that resulted in the disease. Findings revealed
information about disease, which are relevant to the disease diagnosis
and treatment. Several types of mutations have occurred in the gene
one of which was a missense mutation. A missense mutation is a
change in amino acid that causes the protein produced to be nonfunctional. It was learned that hemophilia A has three level of severity
ranging from mild to severe. Based on the sequence analysis data,
there were a lot gaps, which indicate deletions and insertions in the
gene. Since hemophilia A is a hereditary disease a lot of effort is done
to diagnosis it in the early stage. Some novel diagnostics that have
resulted from the knowledge in genetic are clotting factor test,
fibrinogen test, and other revolutionary assays. In an effort to

continuously understand the disease, researchers are working on a

novel method of therapy to insert better functioning factor VIII or factor
IX genes into the cells of individuals with hemophilia allowing their
blood to clot more effectively. Thanks to this new therapy effort, it is
relieving and alleviating to know that this process will lead to patients
having fewer bleeding episodes. In the near future, gene therapies will
likely aid people with hemophilia to begin producing their own clotting
factor, reducing the need for or decreasing the number of weekly
infusions. With this explosion in knowledge, we can help end the
disease for good (12).
Figures

References
1) Http://omim.org/entry/306700#
2) Http://www.ncbi.nlm.nih.gov/books/NBK1404/
3) Http://www.uniprot.org/uniprot/P00451

4) Http://www.ebi.ac.uk/interpro/entry/IPR003245
5) Http://www.ebi.ac.uk/interpro/entry/IPR000421
6) Http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?Rs=137852376
7) Http://pdbj.org/mine/structural_details/1fac
8) Http://www.uniprot.org/uniprot/P12263
9) Http://www.uniprot.org/uniprot/P12374
10)
Http://www.uniprot.org/uniprot/Q700K0
11)
Http://www.uniprot.org/uniprot/P00734
12)
Https://www.hemophilia.org/Bleeding-Disorders/Future-Therapies
13)
Arai, M., Inaba, H., Higuchi, M., Antonarakis, S. E., Kazazian, H.
H., Fujimaki, M., & Hoyer, L. W. (1989). Direct characterization of factor
VIII in plasma: detection of a mutation altering a thrombin cleavage
site (arginine-372----histidine). Proceedings of the National Academy of
Sciences of the United States of America, 86(11), 42774281.