Each cell of the body contains structures called chromosomes. A chromosome is a long chain of chemicals known as DNA. This DNA is arranged into hundreds of units called genes which determine such things as the colour of a person's eyes.
Each cell contains 46 of these chromosomes arranged in 23 pairs, one of which is known as the sex chromosome because it determines the sex of the person. Females have two X chromosomes in this pair; males have one X and one Y.
Figure 3 illustrates the heredity pattern for the sex chromosomes. The mother, who has two X chromosomes, produces an egg containing one X chromosome. The father, who has one X and one Y chromosome, produces sperm which could contain either an X or a Y chromosome. If the father contributes his X, a girl is conceived. If he contributes a Y, a boy is conceived.
The genes responsible for producing factor VIII and IX are situated on the X chromosome. This makes hemophilia a sex-linked genetic disorder.
Figure 4 shows what happens when a hemophiliac male has a child with a normal female.
All the daughters will be obligate
carriers of hemophilia since they must inherit the X chromosome which carries hemophilia from the father. All sons will be unaffected by hemophilia since they inherit the father's normal Y chromosome.
Figure 5 shows the heredity pattern when a normal male has a child with a female carrier of hemophilia. Figure 5
If they have a son, he has a 50% chance of having hemophilia. This is determined by which X chromosome he inherits. If he inherits the mother's normal X chromosome, the boy will not have hemophilia. If he inherits the mother's mutated X chromosome, he will.
In the same way, the couple's daughter has a 50% chance of being a carrier. She could inherit the mother's normal X chromosome, and be normal herself. On the other hand, she could inherit the mother's X chromosome carrying the hemophilia defect and be a carrier.
Because carriers have one normal X chromosome which produces a certain amount of factor VIII or IX clotting factor, they are almost always protected from the most severe form of hemophilia in which the level of clotting factor is less than 1%.
However, the variation in clotting factor levels in carriers is very wide. It ranges from levels similar to those of hemophiliacs in some carriers to normal levels in others. This is because the two X chromosomes, one of which carries the hemophilia gene, are not equally functional. If the hemophilia X chromosome happens to be functional in most cells, then the carrier will have a very low level of clotting factor activity. This is called lyonization, or X-inactivation.
Many carriers have a clotting level between 30% and 70% of normal and do not usually suffer from excessive bleeding. However, some carriers have less than 30% of the normal level of factor VIII or IX. These women are considered to be mild hemophiliacs.
In any case, all carriers should pay close attention to signs of abnormal bleeding. These signs include:
- heavy, prolonged menstrual bleeding (menorrhagia)
- easy bruising
- frequent nose bleeds.
There are two reasons to test a potential carrier:
- to measure the person’s factor level and, if indicated, take precautions to prevent abnormal bleeding
- to know the person’s carrier status.
Different tests are used for each of these purposes.
- Factor level testing - Factor level testing, also called a factor assay, is done to determine a person’s level of factor VIII or IX. This can help determine whether she is at greater than normal risk of bleeding. Such a test does not determine beyond all doubt whether or not a person is a carrier.
- Testing for carrier status - This testing is done to determine whether or not a female is a carrier of hemophilia.
What tests are used, and when these tests can be done are discussed below.
When to test factor levels or carrier status is a controversial subject. The debate centres around whether to test in childhood, specifically before puberty, or to wait until the woman is an adult and can make the decision herself. The issues are different for factor level testing and carrier status testing.Factor level testingReasons for testing in childhood
It is important to know the factor level in a potential carrier due to an increased risk for bleeding that she may experience.
With low factor levels, there is increased risk of bleeding after tooth extraction, trauma or surgery such as a tonsillectomy or adenoidectomy, both common childhood operations.
Because of a surge in the hormones involved to provoke menstruation in young girls, there is a tendency for excessive bleeding at the first few menses, sometimes requiring emergency room treatment. This can be a traumatic event for a young girl and she may be frightened by the amount of blood she loses during her first periods. In addition, almost twice as many carriers as non-carriers require medical intervention for nosebleeds.
All of these potential symptoms suggest the wisdom of early testing to learn clotting factor levels. If levels are low, precautions can be taken.
Guidelines developed by the Association of Hemophilia Clinic Directors of Canada (AHCDC) Sub-committee on Women with Bleeding Disorders recommend that a girl from a family with a history of an inherited bleeding disorder be tested before she starts menstruating. This allows the patient and family to prepare for her first and subsequent menstrual periods, which are sometimes heavy and prolonged.
Some gynaecologists suggest “anticipatory gynaecological management” for young girls. Testing could be done at the first signs of breast development. When possible, investigation should be undertaken before oral contraceptive therapy is begun to control menstrual bleeding.
In addition, if such testing is done, any medical professional who is consulted can be made aware of a carrier’s factor levels. Close collaboration can then be encouraged with the hemophilia treatment team to prevent any medication being prescribed that could affect coagulation or a medical procedure that could cause hemorrhaging.Reasons for delaying testing until adulthood
If a girl has shown no sign of abnormal bleeding (bruising after childhood injury, bleeding after loss of teeth, etc.) parents may choose to wait before testing factor levels. This will avoid a hospital visit and the pain of the blood test. Parents, however, should assume the girl is a potential carrier and take all necessary precautions in the case of surgery and at the onset of puberty.
Being diagnosed with mild hemophilia may affect access to, or the cost of, insurance. Carrier status testing
Reasons for testing in childhood
Early testing allows time for the girl to come to terms with the complex nature of being a carrier. The sooner a girl knows about her carrier status or particular health situation, the sooner she can get the information necessary to help her deal with decisions concerning treatment for any type of medical problem, and eventually with issues of conception, pregnancy and childbirth.
Upon learning of my diagnosis, my reactions went from confusion, fear, sadness and concern to acceptance and hope.
Leaving carrier testing until the girl is in a relationship, or until she is ready to conceive or already pregnant, can create unnecessary stress. Genetic testing for carrier status and genetic counselling is best done before pregnancy. Factor levels rise during pregnancy in the case of factor VIII deficiency, and make assay tests unreliable. Genetic studies take time to carry out. The psychological stress and decisions of prenatal diagnosis can be better dealt with if the woman is not suddenly faced with both the announcement of carrier status as well as the decisions for prenatal diagnosis of the foetus.
In a study done in the United Kingdom with 54 women who are carriers, 83% were in favour of testing for carrier status before the age of 16. The median age that these adult carriers had been genetically tested was 24 years. The fact that most adult carriers in the study believe that carriers should be tested at a much younger age than is currently recommended in the UK (over 16 years) has important implications for the delivery of successful genetic counselling.
If the girl is found not to be a carrier, early testing will have eliminated much stress and concern. There have been cases when a young woman, who assumed she carried the hemophilia gene because it was prevalent in her family, planned her life around the possibility of having a child with hemophilia. She then learned that she wasn’t a carrier and had to readjust to this new reality. Early testing could have saved her from this situation.
Reasons for delaying testing until adulthood
The World Health Organization as part of its hereditary disease program, the American Society of Human Genetics, the American College of Medical Genetics, and the National Society of Genetic Counsellors recommend that, if the medical or psychological benefits from genetic testing will not occur until adulthood, testing should, in general, be deferred until the at-risk female is at an age to understand and make autonomous and informed decisions for herself.
The argument against carrier testing, by DNA analysis, in children is:
- It can harm a child’s self-esteem.
- It can distort the family’s perception of the child.
- It can deprive the girl of the opportunity to make autonomous informed decisions for herself.
- It can adversely affect her capacity to form positive relationships.
Some families tend to delay testing their daughter because they feel the only reason to know about a carrier status is for family planning.
Cultural issues, such as arranged marriages or the possibility of health problems in offspring, may discourage some families from having a daughter tested.
If genetic testing is done in childhood, the girl’s parents are usually the main source of relevant information. If parents misunderstand or don’t remember the test results, the child can be misinformed. Moreover, early testing does not guarantee that parents will inform the child.
In all cases, families should consult their hemophilia treatment team who can help them through the decisional process and with follow-up counselling, if necessary. They will be referred for genetic counselling whose purpose is to provide the carrier and her parents or partner with adequate information to reach decisions regarding carrier testing and prenatal diagnosis, and to provide support throughout the process.
For carriers of hemophilia A and B, the following tests permit accurate diagnoses:
- Factor VIII assay: This measures the amount of factor VIII activity in the blood. Having a normal factor range does not guarantee non-carrier status. Twenty percent of carriers have a normal level.
- VWF: antigen: This measures the amount of von Willebrand factor in the blood so as to rule out von Willebrand disease as the cause of bleeding in hemophilia A.
- Factor IX assay: This measures the amount of factor IX activity in the blood.
- Genetic studies: These tests determine the exact genetic mutation and make it easier to provide prenatal diagnosis. They can identify the mutation in 90% to 99% of cases.
Factor VIII and IX assays
Many elements can affect the results of factor assays. There seems to be a link between hormones and factor levels. Hormone levels in carriers with factor VIII may be affected by a number of factors. Physical and mental stress, recent use of aspirin or other painkillers, pregnancy, contraceptive pills, breast-feeding, exercise, recent transfusions or infections may all affect the results of a factor assay. While it is impossible to avoid all of these influences, they should be taken into consideration. The time to get tested is at the time of menstruation when hormone levels are lowest, though this is still controversial. In the case of factor IX carriers however, clotting levels are not affected by hormones and testing can take place at any time.
Before any coagulation investigation, patients should have a complete personal and family history and physical examination, including a gynaecological examination in most cases (not always necessary in adolescents).Genetic testing
There are a number of tests that can be done to determine carrier status. These tests are complex, not available in all centres and, when available, may take many months to have results. Depending on the set of markers found, the accuracy of these studies varies from 90% to 99 %. Their results are most predictable if DNA from a family member with hemophilia is available.
Women who are identified as obligate carriers or possible carriers of hemophilia (for example, a woman who has already had a son with hemophilia) are eligible for DNA testing. DNA is the building block of genes and can be extracted from a blood sample. DNA testing for hemophilia is covered by provincial health insurance in local facilities. Mutation analysis
Analysis of the hemophilia genes is done by looking for changes in either the factor VIII or factor IX gene responsible for hemophilia. The laboratory begins the analysis by looking for a type of mutation known as an inversion in factor VIII. Half of males with severe hemophilia A have an inversion in their factor VIII gene. If this inversion is not found, then the laboratory needs to do further testing which may be more complex and time-consuming. If a specific mutation is found in the person with hemophilia, accurate carrier testing by mutation analysis can be carried out on the mother or other female relatives who may be carriers. When the mutation can be identified (in 90% to 99% of cases), mutation testing is 100% accurate.
DNA linkage studies
If the mutation is not known, DNA linkage studies are the next step. This involves following markers (or normal variations in the DNA) which either are within and/or surround the hemophilia gene. Linkage analysis is not direct testing such as when a mutation is identified; however, it may be able to provide information about carrier status, with a certain degree of probability. DNA samples from other family members, including those with and without hemophilia are necessary. The male with hemophilia has inherited an X-chromosome with a specific “pattern,” which is responsible for hemophilia in this family, with a certain degree of probability. This pattern may provide information about carrier status.
Hemophilia Treatment Centres are the best choice for comprehensive testing. People at these clinics know that tests often need to be repeated to get an accurate diagnosis. This is because factor level test results can be affected by such elements as a variation in the lab technologist's technique and even the woman’s blood type. (Women with blood type O have naturally lower levels of von Willebrand factor and factor VIII than women with blood types A, B or AB). Many other factors can skew test results. Because of these testing difficulties, many women have been told they have no bleeding disorder when, in fact, they do. Personnel at the HTC are aware of this, and will re-test if a woman continues to have bleeding symptoms. Often, a genetic counsellor or geneticist works very closely with the hemophilia team to provide a multidisciplinary approach to care.
Smaller centres may carry out screening tests including prothrombin time (PT), activated partial thromboplastin time (APTT) and bleeding time, but these tests are not refined enough to detect all bleeding disorders.
Physicians in small centres should refer patients for specialized testing to Hemophilia Treatment Centres. Experts in these centres have the knowledge and experience to do proper testing for carrier status and to deal with the actual treatment of bleeding disorders.