Demystifying Genetic Testing for Bleeding Disorders

When hemophilia and von Willebrand disease (vWD) were first recognized, doctors didn’t diagnose a patient until he (or, much more infrequently, she) experienced a bleeding event that lasted a dangerously long time. But the consequences of the disease had been recognized for centuries: According to the National Hemophilia Foundation, in the second century, the Talmud stated that Jewish baby boys were exempted from circumcision if two of their brothers had already died from excessive bleeding after the process.1

The genetic basis for uncontrolled bleeding episodes was not identified until the early 20th century, when researchers discovered that a hereditary lack of coagulation factors was to blame for abnormal and excessive bleeding. These discoveries led to the development of treatments and prophylaxis that are still used today.

The field made a giant leap forward in the 1980s, when researchers first characterized the genes that encode coagulation factors, opening the doors for genetic testing to aid in the diagnosis and prognosis of patients with hemophilia or vWD.

“Genetic testing results can be very revealing, but we know that genetic testing is still an imperfect and rapidly evolving science,” Stefanie Dugan, MS, CGC, a certified genetic counselor at the Versiti Blood Center of Wisconsin, told ASH Clinical News. “The advances in technology have revolutionized the field, and the clinically available options for genetic testing – not just for hematology, but across different disease areas – have exploded.”

More recently, scientists have set their sights on a more ambitious goal: developing gene therapies to replace defective gene sequences with correct ones, which promises to eliminate disease for a patient’s lifetime.

ASH Clinical News spoke with Ms. Dugan and other geneticists and hematologists about the advances in genetic testing for inherited bleeding disorders, the challenges in the results’ interpretation, and prospects for gene therapies for these conditions.

The Genetic History of Bleeding Disorders

In 1803, a Philadelphia physician, John Conrad Otto, was the first to suggest that bleeding disorders were inherited diseases that primarily affected men and ran in certain families. Twenty-five years later, Friedrich Hopff, a student at the University of Zurich, and his professor Johann Lukas Schönlein, coined the term “haemophilia” to refer to patients who experienced uncontrolled bleeding episodes.

“Genetic testing results can be very revealing, but we know that genetic testing is still an imperfect and rapidly evolving science.”

—Stefanie Dugan, MS, CGC

Researchers also determined that hemophilia is more common among males because the genetic mutations responsible for hemophilia are located on the X chromosome; males have only one X chromosome, so one altered copy of the gene in each cell is enough to cause the condition. However, females have two X chromosomes, so women may carry the mutation without displaying symptoms because their other X chromosome holds a functional gene. Fathers cannot pass X-linked traits to their sons, but a woman who is a carrier has a 50-percent chance of passing the disorder on to a son.

Factor I deficiency was first described in 1920, followed next by the discoveries of factors II and V deficiency in the 1940s. During the 1950s there was an explosion of work on rare factor deficiencies, with scientists describing the role of deficiencies in factors VII, XI, XII, and XIII. This allowed an Argentinian physician, Alfredo Pavlovsky, MD, to identify two types of the disease, hemophilia A (caused by deficient factor VIII [FVIII]) and hemophilia B (caused by deficient factor IX [FIX]).

During the same era, in 1926, Finnish physician Erik von Willebrand described what he called “pseudohemophilia,” a bleeding disorder that affected men and women equally; years later, in 1957, the disease became known as von Willebrand disease, after Inga Marie Nilsson, PhD, and researchers at the Malmo University Hospital in Sweden determined that “pseudo-hemophilia” was caused by a deficiency of a protein in blood plasma that enables hemostasis, later identified as von Willebrand factor (vWF).

Why Use Genetic Testing?

In patients with hemophilia or vWD, a deficiency in one of the coagulation factors due to genetic mutations prevents the body from synthesizing enough functional copies of the necessary clotting proteins. Mutations in FVIII, FIX, or vWF will cause slower clot formation, which can lead to excessive bleeding after cuts and surgeries or bleeding within joints that can cause pain and loss of function.
Genetic testing is the next step after a blood test, like a prothrombin time or partial thromboplastin time test to measure the levels of different clotting factors in the blood.2

“You would never take somebody who is suspected of having, say, hemophilia A and sequence them first,” explained Laura Swystun, PhD, a senior clinical scientist at the Canadian National Inherited Bleeding Disorder Genotyping Laboratory at Queen’s University in Ontario. “You would always first assess their factor activity.”

After conducting a factor assay, a doctor may use a genetic test to confirm the diagnosis and gain more information to guide treatment decisions. Genetic testing “can be a powerful tool to help confirm a diagnosis in someone whose symptoms or laboratory phenotype might be borderline,” said Ms. Dugan.

Also, because hemophilia is inherited in an X-linked recessive pattern, the results of genetic testing have implications beyond the individual patient. “Female carriers of hemophilia can have normal clotting factor plasma levels, and in that case, the only way to identify them would be through genetic testing,” explained Dr. Swystun.

Testing typically finds a disease-causing mutation in up to 98 percent of patients with hemophilia A and up to 99 percent of those with hemophilia B.3 A gene can be rendered dysfunctional by many types of mutations, so tests must do more than identify which gene is mutated. Accordingly, there are a variety of genetic tests than can be performed in patients with bleeding disorders. For most patients with hemophilia A or B, mutations are found in the coding regions of the gene, so that’s where scientists start.

In patients with severe hemophilia A, Dr. Swystun said she usually will first look for inversions – a literal flip or abnormal positioning of the DNA – in introns 1 and 22, which are the most frequently observed mutations in this disease. Inversion analysis can be performed by several different methods of polymerase chain reaction (PCR) or Southern blot analysis. Of the more than 2,000 unique mutations associated with hemophilia A that have been catalogued in genetic databases, most involve point mutations (66.5%), followed by deletions (23%) and duplications (4.8%). These mutations most often occur in the A domains of FVIII.4

More than 1,000 unique mutations have been identified for hemophilia B, and these are present throughout the FIX gene, including the promoter region and the 3’ untranslated region. Like hemophilia A, the majority of FIX variants are point mutations (73%), followed by deletions (16.3%) and insertions (3.3%); however, no inversions have been identified. Mutation analysis typically involves direct sequencing of coding and regulatory regions, although scientists will perform copy number variant (CNV) analysis to determine whether a patient has deletions or duplications of certain genes.

Each testing method carries its own advantages and limitations. Southern blot analysis, for example, is labor-intensive and uses hazardous radiochemicals. PCR can provide results rapidly, but poor amplification of the sample DNA and other errors can produce false-negative and false-positive results.

Unknowns and Inhibitors

And there’s still more to learn, Dr. Swystun said. “There is a small percentage of patients in whom we could sequence everything – their exons, their intron-exon boundaries, the promoter – and we could perform CNV analysis and inversion testing, but we still won’t find out why they have the disorder.”

For these patients, she noted, scientists are exploring newer strategies, like next-generation sequencing, to reveal other variants within deep introns that are causing the disease. “This is still an emerging area for molecular diagnostics,” Dr. Swystun said, adding that “it is important in helping us understand what’s happening with the patient as well as the underlying genetics of the disorder.”

Alternatively, if a patient’s symptoms are severe, a genetic test may help to determine his or her risk of developing inhibitors to certain treatments, explained Bhavya Doshi, MD, a hematologist at the Children’s Hospital of Philadelphia.

Existing treatments for hemophilia or vWD generally help replenish whichever clotting factor a patient is lacking or provide a substitute that performs a similar function to the missing factor. Depending on the severity of the disease, these treatments may be given regularly as a preventive measure or saved for use only in the event of a bleed. The development of antibodies against factors, however, is a common complication of treatment; approximately 30 percent of patients with hemophilia A develop inhibitors to FVIII infusions, making their treatment extremely difficult to manage.5

Several factors, both genetic and environmental, are thought to increase the likelihood that a patient develops inhibitors, but accurate risk prediction remains a challenge. Specific genetic mutations, like FVIII(null), have been associated with inhibitor development; so, if genetic testing reveals that a patient has such a mutation, a clinician can discuss the risk with the patient and his family to decide whether an alternative treatment such as emicizumab (a monoclonal antibody that can substitute for FVIII but doesn’t resemble the protein itself) might be a better option than factor replacement.

Genetic testing also can be a valuable tool in guiding treatment decisions in patients who experience “spontaneous bleeding or who have no family history of bleeding disorders or whose family history is unknown,” Dr. Doshi added. In those instances, she said, “we will – after fighting with insurance – try to get genetic testing done in patients with severe hemophilia [to find out if] this a mutation that’s associated with inhibitors.”

Genetic Testing for All?

Results from genetic tests can reveal important information, but the experts interviewed agreed that clinicians need a better understanding of whom to test – and what to look for. “There is more and more to be learned that sometimes only the genetic testing will reveal. That said, it makes sense to do the right testing for all patients,” Ms. Dugan noted.

The technology used to identify the most common genetic variants involved in hemophilia is readily available to most laboratories, but Ms. Dugan advised that clinicians consult experts in hematologic genetics to ensure that the appropriate tests are ordered and that the results are interpreted correctly.

“It is important that the clinician, as well as the patient and the patient’s family, appreciate what has been tested,” she said. “I have worked with providers who have ordered what they thought was ‘hemophilia genetic testing’ and received a negative result, but they learned later that they had really only ordered one part of the hemophilia testing.” The test may come back negative, she explained, but only for one pathogenic variant associated with that condition. “So, while that part was negative, they actually had to order a different test – the right test – to identify the cause of hemophilia in that family,” she added.

In vWD, there are fewer instances where genetic testing is justified, “because the clinical impact of genotyping in [this disease] hasn’t really been clear,” according to Steven Pipe, MD, director of the coagulation laboratory at Michigan Medicine and chair of the medical and scientific advisory council of the National Hemophilia Foundation. Because the disease-causing vWF gene is so large and contains more than 300 single nucleotide polymorphisms, it is difficult to sequence, and results from genetic testing don’t always provide conclusive answers.6

However, in some situations, genetic testing is justified because its results could aid in genetic counseling or help guide treatment decisions, as with vWD type 3, where identifying certain deletions can predict which patients are at a higher risk for developing neutralizing antibodies. In these situations, mutations are typically clustered in specific areas of the vWF gene, simplifying sequencing and interpretation.

As Dr. Doshi noted, genetic testing is rarely covered by insurance, so advocacy organizations have begun partnering with academic and hemophilia treatment centers to genotype large numbers of patients and grow the genomic database.

For example, in 2012, the American Thrombosis and Hemostasis Network, National Hemophilia Foundation, and Bloodworks Northwest partnered to launch the My Life, Our Future program to offer genetic testing to patients with hemophilia at low or no cost.7 Their stated goal was to create the world’s largest genetic hemophilia repository, which would eventually help scientists answer questions about why the disease’s severity differs widely among patients, who is likely to develop inhibitors, and which genes will be optimal targets for gene therapies. My Life, Our Future participants also can elect to have their genome sequenced through the National Institutes of Health’s National Heart, Lung and Blood Institute’s Trans-Omics for Precision Medicine (TOPMed) Program.8

So far, the program has genotyped nearly 10,000 patients with hemophilia, and Dr. Pipe reported that the program has identified nearly 700 previously unreported variances causative for hemophilia. “This has provided a rich resource for investigators who study molecular mechanisms in hemophilia,” he said, “and they can start to tackle some of the questions about previously unknown mutations.”

The Promise of Gene Therapy

The end goal of collecting genetic sequencing data from a large group of hemophilia patients is to understand the disease fully so that it can be cured on a genetic level. Gene therapy has been on the radar for decades, especially for diseases, like hemophilia, that are caused by a mutation in a single gene; now, researchers are getting closer than ever to that dream.

Methods to “correct” faulty genes using viral vectors have been used in the laboratory in model organisms since the 1950s, but only recently have researchers begun testing them in humans. Currently, there are three gene therapy candidates for hemophilia in phase III clinical trials: valoctocogene roxaparvovec for hemophilia A and AMT 061 and fidanacogene elaparvovec for hemophilia B.

These therapies all rely on engineering recombinant adeno-associated virus (rAAV) vectors to carry a gene of choice to “invade” the genome of another organism. For hemophilia, that means using the technology to deliver functional FVIII and FIX genes to replace defective genes.

Although the progress is promising, few gene therapies actually have been approved for any genetic diseases because there are myriad difficulties in engineering a vector able to carry enough of the functional genes and insert them in the appropriate places at the appropriate times. In early-phase trials, researchers found that many patients had dangerous inflammatory responses to the vectors.

The efforts are further complicated by the complexity of the FVIII and FIX genes. Each is large – too large to fit into the vector. FVIII, the gene that is dysfunctional in the more common hemophilia A, is especially large, so most early advances in gene therapy were made for treatments of hemophilia B. Recently, scientists have found that truncated versions of the genes can fit inside the vectors, and while they may not be as desirable as a fully functional gene, they may be able to improve patients’ symptoms.

For vWD, researchers are adopting a different approach to using genetic technologies. Rather than delivering a fully functional gene, Dr. Swystun described a “workaround” being explored by some scientists. “For example, they now are making portions of the vWF protein that can bind to FVIII independently of the full-length vWF protein, [suggesting] you might be able to treat some patients with this fragment [instead of the whole gene],” he said.

There is still a long way to go before gene therapy finds its way to routine clinical practice. And there are still limitations to gene therapy for inherited disorders; for one, while gene therapies may be able to help patients, they will not prevent them from passing the dysfunctional gene on to their own children in the future. Most investigational therapies have been tested only in adults at this point, and more studies are needed to determine the safety of these treatments in younger patients.

Still, researchers are optimistic. “We should see results from the three phase III trials in the not-so-distant future, hopefully followed by an approval not too much later,” Dr. Doshi said. “I think it’s very close.” —By Emma Yasinski


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  2. Centers for Disease Control and Prevention. “Diagnosis of Hemophilia.” Accessed February 27, 2019, from
  3. National Human Genome Research Institute. “Learning About Hemophilia.” Accessed February 27, 2019, from
  4. Swystun LL, James P. Using genetic diagnostics in hemophilia and von Willebrand disease. Hematology Am Soc Hematol Educ Program. 2015;2015:152-9.
  5. Hemophilia Federation of America. “Inhibitors.” Accessed February 27, 2019, from
  6. Ng C, Motto DG, Di Paola J. Diagnostic approach to von Willebrand disease. Blood. 2015;125:2029-37.
  7. My Life, Our Future. “Research.” Accessed February 27, 2019, from
  8. National Heart, Lung, and Blood Institute. Trans-Omics for Precision Medicine (TOPMed) Program. Accessed February 27, 2019, from