How to Order Genetic Testing for Hemophilia A or B [Profiles in Genetics]

Part 1

 



Identifying the correct test for your patient is always critical. When it comes to hemophilia, there is significant confusion about which tests to order, resulting in about 25% of orders being cancelled after review by Mayo Clinic's genetic counselors. In this two-part presentation, Lea Coon, C.G.C., walks you through the information that you need to correctly select testing for hemophilia A or B. In part 1, Coon provides a thorough review of the appropriate situations when you should test for hemophilia A or B.

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Presenter

Lea Coon, C.G.C., Genetic Counselor in the Hematopathology Laboratory at Mayo Clinic, in Rochester, Minnesota.

Questions?

Contact us: MMLHotTopics@mayo.edu.

 

Transcript

Introduction

Welcome to Mayo Medical Laboratories Profiles in Genetics. These presentations provide short discussion of current genetics topics and may be helpful to you in your practice.

Our speaker for this program is Lea Coon, a Genetic Counselor in the special Coagulation DNA Laboratory at Mayo Clinic, Rochester, Minnesota. In this presentation Lea discusses when to test for, and the appropriate tests for, hemophilia A and B.

Welcome, Lea.

Disclosure

Thank you for the introduction. I have nothing to disclose.

Objectives

The purpose of this presentation is to tell you how to order genetic testing for hemophilia.

Testing for hemophilia is not entirely straightforward, for reasons we’ll get into soon.

I also work in a laboratory that performs this diagnostic testing, and in my experience, about a quarter of the orders we receive for a test specific to severe hemophilia A are cancelled because we find out the test was misordered or misunderstood and it would be wasteful to perform.

Granted, these tests are often misordered for a wide variety of reasons, but in part it is because many people have no idea what they are doing when they are ordering a genetic test for hemophilia.

In this talk and the next, I will cover just 2 things… In the first part of this talk, I’ll go over when it is appropriate to test for hemophilia.

In part 2, I’ll discuss the 2 pieces of information you need to choose the correct genetic test. I’ve simplified this information for anyone who is not a practicing hematologist or geneticist, but a referral to either of these specialized fields should be a consideration when evaluating someone with an apparent bleeding disorder because bleeding is a very nonspecific symptom that can be caused by a great number of things besides hemophilia.

When Should You Consider Genetic Testing For Hemophilia?

But thankfully, there are basically just 2 specific situations in which you should start considering genetic testing for hemophilia. If you don’t have either situation, you probably do not need to order a genetic test for this patient and should consider other options.

Situation 1 is where you have a male patient who is showing signs of soft tissue bleeding and articular hemorrhage, such as joint bleeds, deep-muscle bleeding, bleeding inside the skull, oozing after surgery, and so on.

In severe hemophilia, affected males typically present with these symptoms within the first 2 years of life. In moderate hemophilia, affected males will usually present in their toddler years. Mild hemophiliacs are typically diagnosed later in life and sometimes well into adulthood.

When you see these symptoms, it’s a very good idea to perform some routine coagulation studies to cast the net pretty broadly in your workup, including measurements of prothrombin time (or PT as it is abbreviated), platelet counts, and activated partial thromboplastin time, or aPTT for short.

If PT and platelet counts are normal, but aPPT is prolonged, hemophilia should definitely be a differential, although in some cases, mild hemophiliacs may have a normal aPTT.

Fortunately, some additional screening can be performed to help identify hemophilia and whether it is hemophilia A or hemophilia B. The distinction is very important, as we will discuss.

One of the most important coagulation tests you can perform for suspected hemophilia is measurement of the patient’s factor activity levels, especially factor VIII and factor IX activity levels.

Very briefly, factor VIII and factor IX are proteins that float around in blood, created by the factor VIII or factor IX genes. Although important, we’re not going to review the coagulation cascade here because it is hideously complex.

All you need to know is that these factors are proteins in the blood that help blood clot to stop an injury, and the less functional factor you have, the higher your risk of bleeding to death. And, if you have significantly less of it than what has been observed as normal in humans, that indicates a problem in the gene that produces it.

If the lab work comes back indicating a deficiency of either factor VIII or IX, or less than 40% of what is considered normal for a human, that is the point at which you can begin to seriously consider genetic testing for your patient.

Just a brief aside here, should you have a patient with an apparent deficiency in factor VIII, you will also want to perform vonWillebrand factor antigen and activity levels to rule-out von Willebrand disease, another hereditary bleeding disorder that can mimic mild hemophilia A.

Situation 1 – Affected Male Patient: 3 Important Things to Note

But since we are focusing on hemophilia, you must know 3 important things about genetic testing for male patients.

If your patient does have a deficiency in either of these factors, you pretty much have enough to clinically diagnose him with hemophilia A if he’s got less than 40% factor VIII activity OR with hemophilia B if he’s got less than 40% factor IX activity.

However, although hemophilia can diagnosed based on low factor levels, genetic testing can still be very helpful for confirming the disease and for assessing prognosis and the risk for the development of inhibitors, which are a very grave complication of this disorder.

Additionally, genetic testing can be very helpful for members of his family for all of these reasons listed here:

Genetic testing can be important because not every hemophiliac has the same mutation in the F8 or F9 gene.

And, knowing the specific mutation can make testing easier, less expensive, and more informative for family members.

Also, the identification of the familial mutation is required prior to any prenatal testing on a male fetus at risk for hemophilia.

Just to illustrate, this is just 1 exon in the F9 gene. Each of those red squares represents a specific mutation, either a single nucleotide substitution, or tiny deletions or duplications, etc. As you can see there many, and keep in mind this is only 1 exon out of 8.

So, yes, it would be wonderful if each hemophiliac had the exact same genetic mutation because that would make testing easy, but sadly it is not the case.

The third thing to note about this situation is that before the patient is tested, he should undergo genetic counseling so that he understands the risk, benefits, and limitations of the testing. I hope it goes without saying that no one should ever be forced to or unknowingly undergo genetic testing.

Situation 2 – When Should You Consider Genetic Testing For Hemophilia?

So, that was the first situation in which you should consider genetic testing, and this is the second: When you have a female patient with a confirmed or reported family history of hemophilia, especially if this male is a first-degree relative.

For example, if a woman’s or girl’s father was a confirmed hemophiliac, it is a virtually certainty that she and all of her sisters who share the same father are carriers. Because of that, these women are called obligate carriers.

In fact, if you are able to test this obligate carrier instead of her affected male relative that may in some cases be preferable because you probably want to minimize the number of times you stick your male hemophiliac patient.

If a woman has maternal male relative (like a brother or nephew) who is affected with hemophilia and then has a son with hemophilia, she too is an obligate carrier. Hemophilia is rare enough we wouldn’t consider that coincidental.

However, a woman with no prior family history of hemophilia who has a son with hemophilia may or may not be a carrier. Overall, there is a 2% to 20% chance of her not being a carrier, depending on the type of mutation in the son. This happens sometimes because the hemophilia mutation can arise very early on in the development of the little male embryo, in which case, he’d have the mutation but his mother would not. It’s rare, but it happens.

Other women at risk for carrying hemophilia include those with an affected second- or third-degree male relative, when her mother has not been excluded as a carrier. Although in these cases it is best to test her mom first, because if mom is not a carrier, none of her children will have the mutation either.

Quick Genetics Review

Why is it so important to consider which side of the family – Mom’s side or Dad’s side – has a bleeding issue when we are thinking about hemophilia?

If you are ordering genetic testing, I hope you are familiar with genetic inheritance patterns. In the case of hemophilia, the disease is X-linked, meaning the gene that is mutated in the disease is on the X-chromosome, which men can only inherit from their mother.

Situation 2 – When Should You Consider Genetic Testing For Hemophilia?

Our lab has received orders for hemophilia genetic testing from clinicians who want to evaluate their female patients for hemophilia based on heavy periods and easy bruising, but there is no family history of affected males.

I would like to reassure you that is very unlikely these patients have hemophilia or are carriers of hemophilia. Unless, of course, she happens to have significantly reduced factor VIII or factor IX activity levels.

Why is this?

Abnormally heavy periods can be caused by such a long litany of things, such as hormone imbalance, fibroids, and medications, that a rare inherited bleeding disorder is pretty far down on the list of differentials. And more general bleeding and bruising problems can also be caused by many, many different things such as vitamin K deficiency, liver dysfunction, anemia, etc, that are more common than hemophilia.

Other Bleeding Diathesis Differentials

Even among the inherited causes of bleeding problems, von Willebrand disease, another clotting factor disorder, is more common than either hemophilia A or B, with an estimated prevalence of about 1/100.

And here is a partial list of even more inherited bleeding disorders for your reference.

If you want to address your patient’s concern about a possible bleeding problem in the absence of a family history of hemophilia, there are very informative laboratory screening tests you can use to assess if there is evidence of such a problem, such as looking at prothrombin time or PT, activated partial thromboplastin time (aPTT), platelet count, and factor levels. Any genetic testing ordered prior to performing these screens and the consideration of other, more common, causes of bleeding is premature.

Situation 2 – When Should You Consider Genetic Testing For Hemophilia?

Just one more thing before we move on to the critical pieces of information you need to correctly order genetic testing for hemophilia.

Say you’ve got a patient who reports a family history of hemophilia and you run factor activity level labs and find she is not deficient in either factor VIII or factor IX. Does this mean those labs proved she is not a hemophilia carrier?

Sadly, no.

If you see reduced factor levels, then you can be suspicious for the type of hemophilia associated with that factor. But if you don’t, you cannot rule it out because factor level screening is an unreliable way to detect carriers due to the fact that lots of things influence factor clotting activity in women such as pregnancy, oral contraceptive use, chronic inflammation, blood type in the case of factor VIII, and so on.

This is why genetic testing is important for women at risk for being carriers of a hemophilia mutation, and why such testing is made much easier by first identifying an affected male relative.

That said, the fact that activity levels are unreliable indicators of hemophilia in women should not be seen as a reason to go straight to genetic testing for hemophilia in women with bleeding issues, primarily because hemophilia is a relatively rare cause of abnormal bleeding. In the absence of any family history, these more likely causes should be considered and excluded first before thinking about genetics testing for hemophilia.

Summary

So, to review so far, there are basically 2 situations in which you might want to consider genetic testing for hemophilia.

If you have a patient in mind, and they don’t fit in either of these situations, you may want to consider doing something more useful than performing genetic testing for hemophilia for your patient because it is likely not the problem. If you have a patient who does fit into one of these situations, then please watch for Part 2 of this presentation in early 2017, when we will go over the 2 things you absolutely must know before you order genetic testing for hemophilia.

This concludes Part 1. I appreciate your attention.

Part 2


 

Transcript

Introduction

Disclosure

I have nothing to disclose.

Overview

In part 1 of this talk, I covered when it is appropriate to test for hemophilia. Today, in part 2, I’ll discuss the 2 pieces of information you need to choose the correct genetic test. I’ve simplified this information for anyone who is not a practicing hematologist or geneticist, but a referral to either of these specialized fields should be a consideration when evaluating someone with an apparent bleeding disorder because bleeding is a very nonspecific symptom that can be caused by a great number of things besides hemophilia. You can learn more about the great number of things that can cause abnormal bleeding in part 1 of this talk.

In part 2, we are focusing on the 2 critical pieces of information you need before you start ordering genetic testing for hemophilia.

Things You Need to Know Before Ordering Genetic Testing for Hemphilia

This is thing #1. I know this might seem obvious, but it turns out it is not. Unless the circumstances are pretty extraordinary, you need to know if you are dealing with hemophilia A or hemophilia B, prior to genetic testing.

The purpose of genetic testing is not to help distinguish between hemophilia A and B, because that can be easily and more cheaply accomplished with factor studies in affected males.

“How Do I Tell if I’m Dealing with Hemophilia A or Hemphilia B?”

The purpose is to molecularly confirm the diagnosis and identify the specific mutation causing hemophilia for both prognostic and genetic counseling purposes.

When someone orders genetic testing for both hemophilia A and B on the same patient, at the same time, it indicates that someone somewhere misordered these tests; mistaking them for basic factor clotting activity tests perhaps, or that someone is confused about the use of genetic testing in hemophilia.

And, again, this is how you tell if you are dealing with hemophilia A or B. A deficiency in factor VIII indicates hemophilia A. A deficiency in factor IX indicates hemophilia B.

Things You Should Know Before You Order Genetic Testing for Hemophilia

The second thing you should know prior to ordering genetic testing, particularly if you are testing for hemophilia A, is the severity of the hemophilia in the family in question, which is conveniently based on factor clotting activity.

Speaking in general, and beyond the purposes of genetic testing, this information about clinical severity is important for the patient and the family to know so they have a realistic idea of bleeding risk and prognosis. However, it is also good to know when you are ordering genetic testing for hemophilia, especially in the case of hemophilia A, as we shall see.

Review

So, to review…
You must know 2 things prior to ordering genetic testing for hemophilia.

  1. A vs B
  2. The severity of the disease, which is based on factor levels.

Let’s say you’ve got these 2 pieces of information and that you are dealing with hemophilia B.

If You Are Dealing with Hemophilia B

If, after a thorough evaluation of your patient’s clinical and family history, you find that you are dealing with hemophilia B and the familial mutation has not yet been identified, then you can confidently proceed to full-gene sequencing of the F9 gene.

Full-Gene Sequencing

What does full-gene sequencing mean? Sequencing is the genetic testing methodology used to read the DNA sequence of a gene right down to the base pairs. Sequencing reads the sequence of these base pairs and looks for small alterations in this genetic code that could change the function or expression of the gene.

If You Are Dealing with Hemophilia B

Most mutations that cause hemophilia B are little alterations in genetic code, which is why sequencing will detect pathogenic mutations in 97% to100% of individuals with a clinical diagnosis of hemophilia B.

However, while this method allows us to see small mutations in the DNA sequence, it is too fine grain a method for much larger mutations, such as deletions, that completely eliminate exons, genes, or a part of chromosome, which are also a cause of hemophilia B, but are far more rare than the small mutations we discussed earlier.

Thus, if sequencing is negative or inconclusive, you then move on to deletion and duplication analysis.

Deletion and Duplication Analysis

This testing requires a different method to look for these gross deletions. One such method is MLPA, or multiplex ligation-dependent probe amplification, which uses oligonucleotides, which are short synthetic DNA strands designed to match up with a particular segment of DNA on a gene. This flags whether that segment of DNA is there as it should be or if it has been deleted.

If You Are Dealing with Hemophilia B

So, to review, when you are dealing with hemophilia B, and the familial mutation has not yet been identified, you should order full-gene sequencing of the F9 gene, with reflex to deletion and duplication analysis if sequencing comes back negative or inconclusive.

Once this mutation has been confirmed, other at-risk family members can order testing looking for that specific mutation rather than analyzing the full gene.

If You Are Dealing with Hemophilia A

Now, if you are dealing with hemophilia A and the familial mutation is yet unknown, you definitely need to know the severity of the disease in the family in order to choose the right initial test. Again, severity is based on factor clotting activity in an affected males of the family.

If You Are Dealing with Hemophilia A of Mild or Moderate Severity

If you are dealing with mild or moderate hemophilia A, your job is relatively easy, because you can confidently start with full gene sequencing of the F8 gene and reflex to deletion and duplication analysis if sequencing is negative or inconclusive because, again, most mutations that cause mild or moderate hemophilia A are point mutations detectable by sequencing only.

If You Are Dealing with Severe Hemophilia A

However, if you are dealing with severe hemophilia A, this is where things become a little complicated and strange.

Many times severe hemophilia A is caused by a specific type of mutation that would not be detectable either by regular full-gene sequencing, or standard deletion and duplication analyses.

These recurrent mutations are called inversions.

Inversions

What are inversions? They are basically long stretches of genetic code that are flipped around and inserted back into the chromosome.

Yes, this actually happens.

Inversions—How Does This Happen?

How?

In the case of severe hemophilia A, this happens through a process called intrachromosomal homologous recombination.

Basically, there are stretches of genetic code that lie way outside of the gene that are fairly similar to segments of code inside the gene.

Intrachromosomal Homologous Recombination

During meiosis, the great genomic reshuffling that happens during the creation of eggs and sperm, homologous chromosomes from Mom and Dad are supposed to pair up, intersect with each other or “cross-over,” and recombine to create the unique genome of the germ cell.

But, mistakes can happen. For example, a chromosome can pair up with itself rather than with the homologous chromosome with which it’s supposed to pair.

If that happens, the chromosome recombines with itself. A part of the section of DNA that was located outside of the gene is accidently inserted inside of the gene. The part of the gene this insertion replaces ends up far, far away from its other half. So, this insertion basically rips the gene apart and it becomes completely inactive. There is no factor VIII coming out of that gene ever and no factor VIII levels means this mutation can only cause severe hemophilia A in a male, not moderate or mild.

Now, these inversions are certainly bizarre, but they are also recurrent, meaning these intra- and extrageneic homologous regions near the F8 gene cause enough of a problem during meiosis that we see the mutation pop up time and again.

In fact, most of severe hemophilia A is caused by either an inversion in intron 1 or intron 22. And, because these inversions are not amenable to either full gene sequencing or standard deletion and duplication analysis, if you are dealing with severe hemophilia A, it is recommended you start with a different genetic test specifically designed to detect these inversions.

If You Are Dealing with Severe Hemophilia A

At Mayo, we use a PCR-based inverse shifting method to detect these inversions and we typically test for the intron 1 and intron 22 inversions simultaneously because, together, they account for just over half of all mutations that cause severe hemophilia A. Again, if you are dealing with mild or moderate hemophilia A, you do not have to worry about these inversions.

Why Does This Happen in Hemophilia A Far More Than in Hemophilia B?

We don’t talk about these inversions in hemophilia B because they are very, very rare for that disorder. So why should this happen more frequently in hemophilia A? That’s a good question. It definitely has to do with presence of those disperse homologous sequences in and outside of the F8 gene and likely the location of the F8 gene on the far, far tip of the long arm of the X-chromosome. Since it is at the end, it might be more likely than other parts of the chromosome to fold over and recombine with itself.

If You Are Dealing with Severe Hemophilia A

So, to review, if you are ordering genetic testing for severe hemophilia A, these are the steps we recommend.

Optimizing Genetic Testing For Hemophilia A

Those, my friends, are the basics of ordering genetic testing for hemophilia. If you want to go above and beyond this and be a superstar at ordering genetic testing, you should always submit your patient’s clinical and family history to the lab performing the testing.

Personally, knowing how often these tests are misordered, I become anxious when a client fails to submit patient information to us. Genetic testing is expensive, and the results can be life-changing. Providing the patient information allows us to ensure that the correct test is being performed on your patient.

We welcome the opportunity to discuss your patient and your concerns about selecting the right test with you.

This concludes Part 2. I appreciate your attention.

All future genetic testing topics will be housed within our Hot Topics program moving forward.

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This post was developed by our Education and Technical Publications Team.