A survey of recent discoveries: Identification of an innovative

diagnostic tool for Hemophilia BALS 320: Medical Diagnostics, Fall 2015- Section 1, Team 2

Jesse Forchap, Brandy Fugate, Benjamin Blackwell,

Anthony Park, Caitie Staat, and Khaled Hamad

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Abstract

Hemophilia B is a rare blood clotting disorder that affects an estimated 1 in every 34,000

males in the United States. Hemophilia B is caused by a mutation in the Factor IX gene, which

interferes with the coagulation cascade. The molecular basis of Hemophilia B is complex,

revolving around proper folding of Factor IX such that its activated form can properly participate

in the coagulation cascade. As a result, there are a variety of diagnostic tools used to measure

Factor IX in the blood, but all of these devices are presently used exclusively in professional

point-of-care settings. There has been little effort put forth in the search for an at-home device

that patients can use to monitor and control their coagulation factor levels in the blood. By

presenting a summary of the currently understood pathways, preventions, treatments, and

diagnostic tools involved in Hemophilia B management, we hope to encourage further

investigation into a more convenient diagnostic device and potential new treatment options for

sufferers of Hemophilia B.

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An Introduction to Hemophilia B

Hemophilia B, also known as Christmas disease, is a rare blood clotting disorder caused

by deficiency in the Factor IX blood-clotting factor. The term “Christmas disease” is derived

from the first patient, Stephen Christmas, who was diagnosed with the disease in the early

1950s.1 Factor IX (FIX) is a blood-clotting factor produced in the liver, and is a critical

component of the blood clotting cascade.2 The FIX gene is located on the X chromosome, and

mutations in this gene are the primary cause of Hemophilia B. In some rare cases, the disease

may be acquired during the latter part of a patient’s life as an auto immune disorder. Christmas

disease is the second most common type of Hemophilia; Hemophilia A is most common and is

caused by a deficiency in Factor VIII production.3

Patients with Hemophilia B suffer from joint damage, general organ deterioration, and

abnormal bleeding after minor operations, such as tooth extraction.1 However, the degree of

bleeding varies depending on the individual and the severity of the disease. According to the

National Hemophilia Foundation, there are three main classes of Hemophilia B determined by

FIX plasma levels.4 Severe cases are defined by FIX levels less than 1% of the normal range, in

which patients experience spontaneous bleeding in their joints and muscles. This class of severity

is easily detected through apparent physiological symptoms. FIX levels between 1% - 5% of

normal define moderate cases, and patients in this class generally have bleeding episodes after

injury. Detection of moderate cases is less apparent than severe cases, in that bleeding events are

less apparent. In mild cases, FIX levels are above 5 % but less than 40 % of normal. Mild cases

of Hemophilia B can go undetected for years due to the absence of typical symptoms, as only

severe injury or trauma results in excessive bleeding.

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The FIX gene is located on the long arm of the X chromosome. Females with an FIX

variant on only one X chromosome are considered carriers, but do not express symptoms.2

Female carriers may rarely experience abnormal bleeding episodes after childbirth and require

FIX replacement therapy. 2 Hemophilia B is more prevalent in males because of its X-linked

nature. In the U.S., Hemophilia B occurs in approximately 1 of 34,000 males, and the number of

patients in the U.S. as of 2010 was approximately 4,000. Annual Hemophilia B expenditure in

the U.S. is approximately $58,000 per patient which results in a total of $246.2 million per year

if all patients were to be treated. This cost could increase over the coming years as our

population continues to rise, bringing attention to the importance of Hemophilia B within the

American health care system.5

Current diagnostic methods are primarily quantitative. A variety of these methods will be

discussed in depth. Monitoring of FIX levels comprises the basis of Hemophilia B prognosis.

Presently, FIX levels can only be measured in a professional point-of-care setting, which is

inconvenient to patients. Additionally, treatments for modulation of FIX activity rely on an

approximation of FIX levels in the blood. We hope to encourage additional research by

examining current treatment methods for Hemophilia B and proposing novel diagnostics that

may assist in simplifying treatment and improving quality of life. First, we will delve into current

preventative measures and treatment strategies used to mitigate the symptoms and complications

associated with Hemophilia B.

Preventative Measures and Current Treatment Strategies

While Hemophilia B is rare, it is important that potential parents affected by this disease

examine preventative measures when considering pregnancy. Preimplantation Genetic Diagnosis

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(PGD) is an innovative technique that assists in informing couples prior to conception of the

chances that their child will develop Hemophilia B.6 PGD consists of two main steps: in-vitro

fertilization (IVF) is first utilized, and a biopsy of the embryo is subsequently genetically

analyzed. IVF is accomplished through hyperstimulation of the ovaries followed by retrieval of

the egg, fertilization of the egg, and development of the embryo. Once developed, a small sample

cell is taken from the embryo. Fluorescent In Situ Hybridization (FISH) and Polymerase Chain

Reaction (PCR) are used for genetic evaluation of this sample.6 Embryos that are discovered to

be unaffected by Hemophilia B are then implanted into the mother’s uterus.6 Parents may opt to

conceive naturally and either accept the potential challenges and risks of giving birth to a baby

who has the genetic disorder, or proceed with prenatal diagnosis, then terminate the pregnancy if

Hemophilia B is confirmed.7 However, termination of a viable pregnancy based on a Hemophilia

diagnosis raises strong ethical and cultural concerns.

Hemophilia B is principally treated by managing clotting FIX. Replacement FIX therapy

is the preferred method of treatment for Hemophilia B. Concentrated FIX is regularly injected

into the vein multiple times per week, temporarily replacing the patient’s clotting factor.8

Clotting factor concentrates were previously made via reconstitution of donated human blood.

Donated blood was screened to prevent disease transmission, though risk of contracting an

infectious disease still existed. Clotting factors are now being procured from genetically

modified cells introduced to a hamster cell line. These are known as recombinant clotting

factors.9 The FIX gene is incorporated into the hamster cell genome, and the protein is produced

in large amounts using the cell line. This is less risky because it utilizes animal cells that are free

of human diseases.9 Clotting factors produced through cell lines can be convenient as they have a

long shelf life and can thus be made more readily available to patients.10

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Depending on the severity and pattern of bleeding, there are two classifications of

therapies used. Replacement therapy is used to prevent regular bleeding, and is therefore

prophylactic. Children with severe Hemophilia B use prophylactic therapy on a regular basis.11

There are two sub-types of prophylactic therapy: primary, which young children start using to

lessen or prevent diseases in the joints and will be continued for life, and secondary, which is

used frequently for a limited period of time when bleeding begins.8 The benefits of using

prophylactic therapy include a lower risk of spontaneous bleeding, the ability to participate in

sports, and lower risk of damage to the joints.8,11 The disadvantages of using prophylaxis include

constant injections and inevitably higher expenses.8,11 However, on-demand therapy can be used

in place of prophylaxis for the purpose of occasional or sporadic bleeding, as needed. On-

demand therapy is less rigorous and less expensive than prophylactic therapy. 8

One of the most severe complications that can develop through treatment of Hemophilia

B is antibody resistance to clotting factor.12 FIX can be destroyed by these antibodies, which then

prevents replacement therapy from working properly. These antibodies are known as inhibitors,

and they develop in 1.5-3% of Hemophilia B patients.8,12 Figure 1 depicts the Bethesda assay

used to detect the inhibitor concentration (or amount of antibodies) in the blood.8

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Figure 1: Bethesda units (BUs) and factor present in blood post-injection. A higher BU value indicates a

higher concentration of inhibitors present in the blood, which results in less active FIX post-injection.

When the inhibitor concentration increases, additional FIX must be injected into the patient to maintain

healthy FIX levels. Adapted from www.sevensecure.com/inhibitor/inhibitor-education.aspx

Stemming from our conversation on current treatments, we will now discuss the basis of

Hemophilia B on a molecular level, and how the various blood-clotting factors play their roles in

the clotting cascade.

The Molecular Pathway of Hemophilia B

The pathway of Hemophilia B was first elucidated in 1986 by Diuguid, et al. at the New

England Medical Center and Tufts University School of Medicine in Boston.2 The FIX protein is

synthesized exclusively in hepatocytes and is produced as a zymogen, or an inactive precursor.2,13

In the unadulterated pathway, FIX is first translated by ribosomes in hepatocytes, and is

structured as a single chain plasma glycoprotein peptide sequence.14 The FIX molecule is

comprised of three main units: the signal peptide, which facilitates the transport of the protein

across the plasma membrane, the propeptide sequence, and the final protein product.2 Before any

posttranslational modifications are made, the signal peptide is first cleaved from the FIX

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molecule once it has exited the nucleus. Next, a series of modifications occur including

glycosylation, cleavage of the propeptide, beta-hydroxylation, and vitamin K-dependent

carboxylation.2,14 Once these posttranslational modification reactions are completed, the FIX

zymogen is released from the hepatocyte.

It is important to note that this pathway occurs on the platelet surface at the site of

damage along a blood vessel.15 Effective completion of the blood coagulation cascade can only

occur on cell surface membranes.16 In order for FIX to be activated, it must be converted into a

serine protease by Factor XIa in the presence of Ca 2+ .14 When FIX experiences these conditions,

the hydrolysis of two peptide bonds buried inside the Factor IX molecule occurs, which allows

for the release of the activation peptide.14 This results in procurement of Factor IXaβ, which is the

active and final form of FIX. It is comprised of a single heavy chain and a single light chain,

with the heavy chain containing the active site.14 Factor IXaβ activates Factor X along with

assistance from Factor VIIIa.16 This occurs though complexing of Factor IXaβ and Factor VIIIa on

the membrane surface, whose new product is able to generate Factor Xa.16 Factor Xa then

complexes with Factor Va, which promotes activation of prothrombin. Finishing out the pathway,

the now active thrombin cleaves fibrinogen present near the platelet surface, which then

polymerizes to form a fibrin clot.16 Figure 2 illustrates the previously described pathway.

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Figure 2: A display of the entire coagulation cascade from initial injury to final clot formation. This figure

shows all key players involved in the formation of a clot. Adapted from the Hemophilia Report 2014.6

There are two types of defects that have been characterized in Hemophilia B:

independent deletion mutations and point mutations.16 This will be discussed in depth in the next

section. For the sake of simplicity we will discuss only one of the many possible mutations that

leads to Hemophilia B that disrupts γ-carboxylation. However, this discussion is linked to a

broader context in that many mutations that cause Hemophilia B interfere with γ-carboxylation.16

FIX is vitamin K-dependent, meaning that it requires a carboxylase to bind to it in the presence

of vitamin K in order to become secreted. In the point mutation being discussed, an arginine

becomes a serine at the -1 residue, as discussed in the article by Diuguid, et al. This leads to

interference of γ-carboxylation, and ultimately causes failure of the cleavage of FIX’s

propeptide.2 FIX subsequently misfolds and cannot activate Factor X because it is incapable of

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complexing with Factor XIa on the cell membrane. From this point, Factor X cannot go on to

promote activation of prothrombin. Thus, the clot cascade fails and excessive bleeding occurs at

the site of injury.

This mutation example can be extended to some other deletion mutations that cause a

similar non-recognition of the FIX molecule by the Vitamin K-dependent kinase within

hepatocytes. From the elucidated pathway of Factor IX, and subsequently Hemophilia B, we can

derive some key biomarkers that can be utilized in the diagnosis and monitoring of this disease.

Genetics Research and Biomarkers of Hemophilia B

Hemophilia B is caused by mutations in the Factor IX gene, and there are more than

1,100 such mutations that have been identified.17 The most common FIX mutation that causes

Hemophilia B is substitution of a single DNA base pair, although other mutations may still

occur.18 These mutations are linked to a decreased quantity of active FIX and lead to defective

blood clotting and excessive bleeding. Therefore, severity of Hemophilia B chiefly depends on

the severity of the mutation.

Identification of FIX mutations within their locus on the X chromosome is essential

because identified regions can be used as genetic markers to treat Hemophilia B. Two common

types of genetic screens performed to identify heterogeneous mutations in FIX are Restriction

Fragment Length Polymorphism (RFLP) analysis and haplotyping. RFLP analysis is a technique

that exploits variations in homologous DNA sequences. A haplotype is a group of genes within

an organism that were inherited together from a single parent. Both of these analyses can offer

prevention through screening by detecting X-linked gene carriers.

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Figure 3 Informativeness of the RFLP markers in different population groups of India.19 Panels A, B and

C respectively represent the Eastern, Southern and Western regions. RFLP markers analyzed in each bar

diagram are denoted by DdeI (D), HhaI (Hh), Hpy188I (Hp), MnlI (M), TaqI (T), and XmnI (X).19 Panel

D shows informativeness for North India obtained from a separate study (Chowdhury et al. 2001).19

Informativeness for each marker was calculated as the percentage of females heterozygous in each

cluster.19 Adapted from Mukherjee, S. et al., 2006.19

For RFLP analysis, Mukherjee, et al. collected data from eight different populations that

were composed of 107 normal females from different geographical areas with clear family

histories of Hemophilia B, and 13 carriers that were unrelated to each other.19 Then, DdeI, XmnI,

MnlI, TaqI, and HhaI restriction sites were used to identify the genetic markers (Figure 3).19 In

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addition to RFLP analysis, researchers also used Single Nucleotide Polymorphisms (SNPs) to

screen regions of the FIX gene.19 The results from this study on various populations in India

showed that only two SNPs were found as possible candidates for differentiating normal groups

from carrier groups, whereas RFLP markers on specific groups were effective in differentiating

between carrier groups and normal groups.19 The study also mentioned that additional genetic

markers were necessary for more efficient analysis because the variability of heterozygosity was

quite high.19

Another study done in Sweden focused on discovering the origin of high occurrences of

certain FIX mutations in specific subgroups of the population.17 Their objective was to analyze

and classify FIX mutations within Swedish families into two different types of mutations. These

were classified as independent recurrent mutations (RMs) or common mutation events, also

known as identical by descent (IBD).17 By resequencing and performing haplotype analyses on

86 Swedish families with 74 genetic markers, researchers found that both RMs and IBDs were

present at the same proportion -slightly over 50%- in patients with the mild severity phenotype

of hemophilia B.17

Genetic marker analyses of the FIX gene on different population groups from different

geographical regions showed a wide range of variability and specificity based on types of

analysis performed. In juxtaposition to the RFLP markers used in the study performed by

Mukherjee, et al. (Markers 20-30), the study performed in Sweden used the M1 marker.20 Since

genetic markers vary greatly from population to population, discovery of new genetic markers by

performing different experimental methods such as Random Amplified Polymorphic DNA

(RAPD), Amplified Fragment Length Polymorphism (AFLP) and Quantitative Trait Locus

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(QTL) analyses could potentially lead to more efficacious and specified genetic markers of

Hemophilia B.21

Coagulation FIX, translated from the FIX gene, is the most well-researched biomarker

used for detecting Hemophilia B.18 FIX is associated with an activated partial thromboplastin

time (aPTT) of the intrinsic pathway.22 As was stated before, in the normal coagulation cascade,

FIX is activated by Factor XIa and activates Factor X with assistance from Factor VIIIa and other

molecules.23 Although levels of Factor XI, Factor X and Factor VIII could be possible candidates

for alternative biomarkers of Hemophilia B, FIX is still strongly recommended since other

factors are placed either upstream or downstream of the FIX activation within coagulation

pathway. Therefore, FIX is the most logical biomarker to utilize for Hemophilia B at the present

time. We will now move into the diagnostic methods that use FIX to identify Hemophilia B.

Diagnostics for Hemophilia B

Various types of tests have been implemented for screening and diagnosis of Hemophilia

B, though quantitative tests are predominantly employed in regular practice. The Hemochron®

Signature Elite, a handheld point-of-care testing device, can perform various coagulation tests

and is commonly used in clinical settings.24 For initial screening, activated partial thromboplastin

time (aPTT) is most often utilized, and is a one-stage clotting assay.25 Both aPTT and an

alternative screening coagulation test, prothrombin time (PT), can be determined by the

Hemochron® system.24 Both tests are performed using similar mechanisms, but each requires

different sample types and reagents.

Sample separation is not required in the Hemochron® system, as this device is a whole-

blood system. Venous or finger-stick samples are analyzed.24 Testing occurs within disposable

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cuvettes preloaded with reagents; these reagents increase the sensitivity of each assay.26,27 PT

sample tubes are preloaded with dried thromboplastin,26 whereas sample tubes for aPTT testing

are preloaded with platelet factor substitutes and kaolin reagents that standardize the activation

of clotting factors.27 Respective stabilizers and buffers are also included in both types of sample

tubes.

When a sample is introduced to the system, the device dispenses 15 microliters of that

sample into a cuvette. The device automatically performs reagent mixing. An incubation period

is not required for aPTT testing.27 As the cuvette is moved rapidly back and forth within the

instrument, motion of the sample is monitored and measured by LED optical detectors in line

with the sample. As the sample clots, the overall motion of the sample is reduced. When the

movement has decreased below a certain threshold, the device identifies that an endpoint has

been reached. The time required to clot is reported respectively for each test. Results of both tests

can be obtained in approximately two minutes.26,27 PT results are represented in terms of the

International Normalized Ratio (INR), a ratio of the patient’s resulting PT compared to a normal

range that is designated internationally.26 Activated PTT results are expressed in terms of their

plasma-equivalent values, which are automatically calculated by the system.27

The primary test currently administered for final diagnosis of Hemophilia B is a FIX

assay. One type of analyzer developed for coagulation automation utilizes electromagnetic

mechanical detection methods to detect plasma levels of activated FIX.28 Siemens has developed

one such analyzer called the Sysmex® CS-5100 System. Although this device is not currently

available in the U.S., other devices that employ this type of technology are commonly

utilized.28,29 The Sysmex® System, similar to the Hemochron® device, measures motion within

the sample provided.29 FIX deficient plasma is the primary reagent added to the sample plasma in

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these assays.30 Factors within specimens given by healthy patients are able to correct for deficient

factors within the reagent, and clotting will occur. However, in Hemophilia B patients, clotting

will not occur as both the reagent and sample plasma have factor deficiencies.31 Fibrin formation

is measured as it surrounds an iron ball that is also added to the plasma.32 Electromagnetic action

moves the iron ball within the plasma. Mobility of the ball is reduced as a clot accumulates

around it. The device detects the consequent lack of movement and provides an output value.32

Final diagnosis of Hemophilia B can be assigned a level of severity: mild, moderate, or severe.4

Disease severity along with associated signs and symptoms is outlined in Table 1.

Table 1: Clinically assigned severity of Hemophilia B.

Severity Factor IX levels Relative signs of deficiency

Mild >5% FIXa activity in plasma Prolonged bleeding post-surgery

Moderate 1% – 5% FIX activity in plasma Excessive bleeding after injury; occasionally spontaneous bleeding

Severe <1% FIX activity in plasma Frequent spontaneous bleeding episodes

Clinically assigned severity of Hemophilia B is listed according to incremental levels of Factor IX

activitya.8 Various physiological signs may also correspond with the severity of the disease.

FIX is a reliable biomarker that is specific to Hemophilia B. However, diagnostics are

presently only available in professional settings, which may be due to rarity of the disease and

the resultantly small market size. Development of a portable device designed to measure

activated FIX levels would benefit Hemophilia B patients, as the availability of a point-of-care

diagnostic tool is a critically unmet need. Many patients with mild or moderate disease diagnoses

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may not be aware of less obvious internal bleeding, placing them at risk for development of

comorbidities such as hemarthroses (bleeding in joints), renal disease, and cardiovascular

complications.8 Routine, prophylactic treatment for management of bleeding reduces the

likelihood of developing these conditions.8 Rapid testing methods could therefore assist in

reducing the occurrence of associated diseases.

Further research should be done to determine whether or not an at-home diagnostic

device could be applied to routine patient care, thereby reducing the long-term side effects of

bleeding. Traditional assay techniques may be utilized to quickly measure levels of FIX in the

blood. This would assist a patient in determining when treatment is necessary. Some patients

naturally develop inhibitors against FIX added to the bloodstream,12 indicating that development

of an immunoassay might be an effective way to construct a new diagnostic tool. Potential

integration of a color change mechanism might be utilized to determine how much of the analyte

has bound to a test line. This could provide a partially quantitative result, allowing patients to

identify when immediate injections of FIX replacement are required.

Looking Ahead: Future Treatments of Hemophilia B

Patients suffering from Hemophilia B are currently unable to measure the

concentration of FIX at home. Current available options include waiting for physiological

symptoms to arise, which can increase the chances of unnecessary bodily harm, or to inject FIX

multiple times a week and assume that levels of FIX are stable. A new possible delivery method

that can solve these issues is an implantable closed loop device, which has proven successful in

diabetic patients.

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Figure 4 Implantable Glucose Device33 retrieved from Implantable closed-loop glucose-sensing and

insulin delivery. Adapted from Renard E., 2002. 33

The glucose device shown in Figure 4 stabilizes patient’s blood glucose levels over

longer periods of time compared to regular insulin injections.33 This device contains an

intravenous glucose sensor, compartment to store insulin, and a catheter. The glucose sensor uses

an enzymatic reaction involving glucose-oxidase that is monitored by an oxygen sensor; the

sensor measures changes in oxygen levels, and sends a signal through a subcutaneous connector

that relays signals from the sensor to the compartment filled with insulin.33 The device then uses

a mathematical algorithm to release a specific concentration of insulin through the catheter33.

Implementing a similar device into a Hemophilia B patient’s treatment regimen would reduce

their risk of joint degradation or other associated diseases by maintaining FIX levels in the blood.

However, there is currently no sensor on the market that can measure FIX in solution.

One way to improve quality of life of Hemophilia B patients is by reducing the number of

injections needed per week. It has been proven that PEGylation of certain proteins can increase

the half-life of a protein in solution. PEGylation is performed by covalently binding a

polyethylene glycol molecule to the desired protein.34 The addition of polyethylene glycol

External Programmer using telemetry

Central port refill

Intraperitoneal catheter

Abdominal connecting lead (subcutaneous)

Subcutaneous connector

Intravenous enzymatic glucose sensor

Slideport

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decreases the chances of protein metabolism, propensity for immunogenic response, and

clearance.34 By PEGylating FIX concentrates, we can expect to see a decrease in the number of

injections per week, and a reduction in the immunogenic response against FIX. However,

PEGylation has some potential side effects. PEGylation has increased coagulation of proteins in

some cases, which increases the chances of clot formation in blood vessels, causing thrombosis.34

Further studies should be conducted to see the effects PEGylation may have on FIX coagulation

in particular.

An alternative treatment to injecting FIX concentrates is gene therapy. Gene therapy

provides a way of introducing a wildtype FIX gene into a cell to be expressed if the host gene is

mutated or defective. The gene can either be integrated directly into a cell’s chromosome, having

a long-lasting effect, or can be delivered to the nucleus, which has a short-lasting effect because

the gene is attached to a free-floating plasmid.35 The type of delivery method will therefore

predict whether the gene therapy will result in a treatment or a cure. There are three possible

methods that could be used to deliver the FIX gene to its target: adeno-associated vectors, naked

plasmids, and lentiviral vectors. By using adeno-associated virus vectors (AAVs), the FIX gene

can be inserted inside a virus that delivers the FIX gene to the nucleus where the gene is

expressed by the host’s cellular machinery (Figure 5).13 Unfortunately, there have been instances

of immunogenicity occurring using this method, as the virus is identified as non-self by the host

immune system.35

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Figure 5 Gene therapy using Adeno-associated virus vector. Adapted from Carr, et al. 2015. 13

Utilization of naked plasmids is an alternative approach to avoiding immunogenic

response since they are a non-viral vector, although this method has a less permanent effect than

AAV. Naked plasmids cannot insert the gene into the host cell’s target chromosome, so the

expression of Factor IX will fade with time. It is also difficult to target specific cells using this

method.1

Lentiviral vectors can incorporate FIX gene into the host cell, which means FIX

expression would not fade over time.35 Lentiviral vectors have been used in both in-vivo and ex-

vivo studies. Utilization of a lentiviral vector produced an immunogenic response similar to the

AAV vector in-vivo.35 However, when the lentivirus was transduced via ex-vivo techniques it did

not initiate an immunogenic response.35 Thus, ex-vivo transduction of FIX gene may be a

potential treatment for Hemophilia B in the future.

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Conclusion

While the cause of Hemophilia B has been known since the mid-1950s and treatments

have subsequently been produced, there has yet to be any significant development of an at-home

diagnostic tool to improve patient quality of life. The multitude of diagnostic tools that have thus

far been produced are exclusively purposed for professional point of care or lab-based settings.

We hope that from the information and suggestions put forth in this paper, further research may

be performed to develop an at-home diagnostic tool to assist patients in maintaining persistent,

healthy FIX blood levels.

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