Hemophilia A, an X-linked recessive genetic disorder and one of the most common human coagulopathies, occurs when there is a shortage of the clotting factor known as factor VIII (FVIII) and affects approximately 1 in 10,000 men.
The gene mutation is present in both men and women and in approximately 85% of sporadic cases, the mother is a carrier. The remaining 15% of non-carrier women are still at risk for germinal mosaicism.
The FVIII molecule forms a non-covalent bond with a multimeric glycoprotein called von Willebrand factor (vWf), which stabilizes the activity of FVIII and transports it to damaged tissue during hemostasis. A lack or shortage of FVIII alters the effects of this hemostatic process. The clinical severity of hemophilia A is directly related to plasma FVIII concentration, whereby levels below 1% indicate severe hemophilia, 2 - 5% indicates moderate hemophilia and 6 –20% indicates the mild type. Patients with a severe deficiency of FVIII may suffer spontaneous intra-articular bleeding (hemarthrosis), especially in the knees, ankles, wrists, elbows and hips, giving rise to progressive arthropathies if left untreated. Intra-muscular and retroperitoneal hemorrhaging can also occur.
Even though hemophilia was considered a fatal disease, replacement therapy (with either blood derivatives or FVIII:C concentrates) has meant a huge advance in terms of life expectancy and quality of life. However, until the use of viral inactivation and recombinant FVIII:C concentrate methods, replacement therapy implied a risk of transmitting other diseases such as hepatitis and HIV. Inhibitors that work against FVIII:C currently constitute one of the most prominent therapeutic problems. In about 15% of hemophilia A cases, the patients’ bodies produce FVIII antibodies whose inhibitory activity works in response to the FVIII infusion. In the past, carriers were determined based on Bayesian analysis of both the family tree and the amount of active FVIII, vWf and ristocetin cofactor in the blood.
This analysis made it possible to predict the genotype for 70 - 95% of potential carriers. Additionally, prenatal diagnosis used to depend on measuring the clotting factors present in fetal plasma using fetoscopy and later, cordocentesis. However, identification of the FVIII gene has made it easier to do prenatal diagnosis and linkage analysis in carrier screenings. Molecular pathology of hemophilia A
The gene coding for factor VIII, one of the largest known genes (186Kb), was cloned and characterized in 1984 and found to have very heterogeneous mutations.
In 1993, the most common mutation that affects approximately 45% of hemophilia A families was identified as being a homologous recombination between the intron 22 sequences and other sequences on a telomere of the X chromosome. This rearrangement causes part of the gene’s 3’ end to invert and transcription to cease. In other words, the gene splits in two and remains inverted and separated by 500 Kb. A direct analysis of the mutation can be done to detect other inversions. Patients whose gene is not inverted are tested for other gene mutations using intragenic and extragenic DNA markers.
These markers are also used in a highly reliable linkage analysis carried out to determine obligate and potential carriers. Carrier screening and genetic counseling Analysis entails carrier screening for women with a family history of hemophilia A and prenatal diagnosis for hemophilia A. The following analyses can be carried out: Direct analysis of the mutation: Southern blot analysis using the restriction enzyme Bcl I and the probe 482.6 is carried out to identify FVIII gene inversions (PCR detects inversion, but does not indicate if it is distal or proximal). Selective amplification of the coding regions of the factor VIII gene, SSCP and sequencing are used to detect other mutations in patients with no FVIII gene inversion.
Genetic linkage analysis: As it is impossible to detect all mutations, intragenic and extragenic markers from the FVIII gene are used to indirectly analyze the presence of the defective gene. These markers have two intragenic restriction polymorphisms (intron 18 Bcl I and intron 22 Xbal), two intragenic (CA) microsatellites (intron 13 and 22) and two extragenic markers (St14 and Dx13). Either Southern blot or PCR analysis is used to detect these polymorphisms. Samples from different family members including parents, grandparents, potential carriers and those affected are needed to carry out the examination.
2. (Fedhemo #27 pgs 7-31)
3. Tizzano E. , Domènech M., Altisent C., Tusell J., Baiget M: Inversions In the Factor VIII Gene In Spanish Hemophilia A Patients. Blood 83:3826, 1994.
4. Tizzano E., Domènech M., Baiget M. Inversion of the Intron 22 In Isolated Cases of Hemophilia A. Thrombosis and Haemostasia 73:6-9 (1995).
5. Antonarakis SE et. al.: Factor VIII Inversions In Severe Hemophilia A: Results of An International Consortium study. Blood, 86:2206-2212, 1995.
Hemophilia B, also an X-linked genetic disorder, results from a deficiency or malfunction of the vitamin-K- dependent clotting factor, factor IX (FIX). Although less common than hemophilia A, affecting approximately 1 out of every 30,000 males, the clinical-pathological, social and economic importance of this disease is comparable to that of hemophilia A.
The age at which hemophilia B patients start experiencing bleeding episodes is related to their factor IX plasma levels, whereby factor IX activity less than 1% corresponds to severe hemophilia B and between 2 – 5% and 5 – 30% corresponds to moderate and mild hemophilia B, respectively. Molecular pathology of hemophilia B Hemophilia A and B share many of the same molecular mechanisms responsible for their clinical expression.
However, unlike the intron 22 inversion in hemophilia A, there is no principal mutation in hemophilia B. Current research shows that mutations of the factor IX gene (34Kb) are also heterogeneous, making it difficult to identify them. Intragenic and extragenic DNA markers can be used to indirectly study the anomalous segregation pattern in families affected with hemophilia B.
However, some families may lack informative DNA markers. The intragenic markers are found in the gene and the extragenic markers are close to the gene in the q28 region of the X chromosome. Using these two types of markers minimizes the probability of recombination between the mutation and the analyzed polymorphism(s), which yields highly reliable, but probabilistic results. Carrier screening and genetic counseling The analysis entails a carrier screening of women with a family history of hemophilia B and prenatal diagnosis of hemophilia B.
The following tests can be carried out: Direct analysis of the mutation: Selective amplification of the coding regions, SSCP and sequencing are used to detect mutations. Genetic linkage (indirect) analysis: Intragenic and extragenic markers from the factor IX gene are used to indirectly analyze the presence of the hemophilia gene.
These markers include five intragenic polymorphisms: intron 1 DdeI; intron 3 XmnI; intron 4 TagI; 3’ HhaI. Uninformative cases are analyzed using two flanked extragenic microsatellites: 5’ 1192 (DXS102) and 3’ 1205 (DXS105). Detection of these polymorphisms is carried out using PCR analysis. Samples from different family members including parents, grandparents, possible carriers and those affected are needed to carry out the examination.
2. Peak IR, Lillicrap DP, Boulyjenkov V, Briët E, Chan V, Ginter EK, Kraus EM, Ljung R, Manucci PM, Nicolaides K, Tuddenham GD. Report of a Joint WHO/WFH Meeting On the Control of Haemophilia: Carrier Detection and Prenatal Diagnosis. Blood Coagulation and Fibrinolysis, 4:313-344,1993.
3. Gianelli F. y Green P. The Molecular Basis of Haemophilia A and B. Baillière´s Clinical Hematology, 9:211-228, 1996.
4. Montejo JM, Magallón M, Tizzano E, Solera J. Identification of Twenty-one New Mutations in the Factor IX Gene By SSCP Analysis. Human Mutation 13:160-165, 1999.
The Genetic Laboratory of Hospital Sant Pau is Spain’s main center for the molecular study of hemophilia A and B. This group received the 1999-2000 DUQUESA DE SORIA award for its work published in the following article: MOLECULAR PATHOLOGY OF THE FACTOR VIII GENE: ITS RELATION WITH INHIBITOR DEVELOPMENT, ITS APPLICATION IN GENETIC COUNSELING AND ITS IMPORTANCE IN NEW THERAPEUTIC ALTERNATIVES (Fedhemo No. 27).
Of the 305 families studied by the research group, 163 had a family history of hemophilia A and 142 presented with sporadic cases. Severe, moderate and mild hemophilia A was found in 197, 64 and 44 of these families, respectively.
The factor VIII mutation was detected in 133 families, from which 99 obligate carriers and 282 potential female carriers were analyzed in order to determine whether they carried the mutated gene.
The mothers were analyzed in 15 families in which an affected member was not available. The families were divided into three groups: 58 with a family history (more than one affected person in two generations); 8 with possible mosaicism (more than one affected person in a sibling generation, but with no other affected individuals in the family); and 67 sporadic cases.
The direct and indirect molecular diagnoses of all the cases studied are summarized in Table I (Fedhemo No. 27, pgs 7-31) The carrier studies and analyses of family-history and sporadic cases can be found in Table IV (Fedhemo No. 27, pgs 7-31) Prenatal diagnosis of hemophilia A.
The detection of mutations made it easier to do prenatal diagnoses on 38 female carriers for hemophilia A, some of whom had requested the diagnosis on more than one occasion. In all cases there was correlation between the indirect analysis (the polymorphisms) and the direct analysis.
The conclusions drawn from the research carried out by this laboratory give us a good idea of how to do a good molecular analysis of this disease. In addition, the large size of the sample group analyzed means these conclusions offer very reliable information. Conclusions (Fedhemo No. 27, pg. 29)
Tables I and IV
Immunology’s Molecular Biology Division (Dr. de Juan) collaborates with Hematology (Dr. Maite Uranga), offering technical support for the genetic analysis of patients with and carriers of hemophilia A or B and their family members.
The aforementioned division elaborates family tree charts, determines which carriers and family members to study in each case, sends processed samples to be studied to Hospital Sant Pau’s Genetics Unit and acts as a spokesperson during the entire diagnostic process.
Since the start of this collaborative effort in 1997, the following cases have been analyzed: 12 families with hemophilia A, half of which had a family history and the other half of which presented with sporadic cases. 4 of these families had severe hemophilia A , 3 suffered the moderate type and the remaining 5 had mild hemophilia A.
A total of 13 obligate carriers and 38 potential carriers were analyzed.
The study of 10 of the families was informative and the results from one of the remaining two are still being processed. Three FVIII gene mutations were characterized and molecular analysis showed intron 22 inversion. A pregnant woman was given a carrier screening test and prenatal diagnosis was carried out. 3 families with hemophilia B were analyzed, 2 presenting with sporadic cases and one with a family history. There were 3 obligate carriers and 12 potential carriers and all three cases proved to be informative. Prenatal diagnosis was carried out.
Genetic counseling is an educational and informative process designed to treat problems related to the appearance or risk of recurrence of a specific disorder within a family. In our case, this disorder is hemophilia.
1. Help inform people of their own well-being and that of their offspring.
2. Guarantee informed consent for carrying out genetic tests and explain the procedures used, being sure to include information about the following:
hemophilia, prognosis and therapy options
mode of transmission
hemophilia and carrier diagnostic tests and their reliability
carrier screening procedures
prenatal diagnosis, tests and risks for both the mother and fetus
information regarding test results
ways of terminating pregnancy and related future implications.
3. Teach hemophilic patients how to talk about their condition and how to inform family members about the hereditary risks. It is important these patients be supported in any decisions they make.
4. Provide couples and families with the opportunity to attend sessions organized by Hematology or the Hemophilia Association.
5. Find out how it was transmitted. Be sensitive when talking to people of different religious, cultural, social and personal backgrounds.
In summary, the family seeking treatment should know what the disease is, how it is treated, its mode of inheritance, the likelihood their offspring inherit it, error factors in risk calculation and diagnostic testing, etc. They should also be informed of alternatives that exist so they can choose the best treatment plan. Elaboration of a detailed family tree with reliable information is very important. Errors in diagnosis or genealogy information can be misleading when interpreting results.
Hemophilia A and B are the most severe genetic bleeding disorders in humans, the former accounting for about 80% of all hemophilia cases. The incidence of hemophilia A is 1-2 out of every 10,000 live births, whereas that of hemophilia B is 2-4 out of every 100,000. It is estimated 1 in 5,000 women is a carrier in the general population. Hemophilia A results from a deficiency or abnormality in active clotting factor VIII (FVIII:C), a glucoprotein of low molecular weight that is responsible for coagulation. FVIII:C binds to a carrier molecule of high molecular weight known as von Willebrand factor (VIII:Ag). These two coagulation factors are normally found in equal quantities. While FVIII:C, whose deficiency causes hemophilia A, is coded by a gene linked to the X chromosome, FVIII:Ag, whose deficiency causes von Willebrand disease, is coded by an autosomal gene found on chromosome 12.
Hemophilia B, also known as Christmas disease, occurs when there is a shortage of or abnormality in a vitamin-K-dependent clotting factor known as factor IX (FIX). The social, economic and clinical-pathological importance of both hemophilia types is well known and their severity is related to blood levels of their respective deficient clotting factors. Levels between 0-1% is considered severe, whereby the patient experiences frequent and spontaneous bleeding episodes. Levels between 1-5% is generally classified as moderate, although in some cases clinical manifestations appear to be those of the severe type. Levels between 5-30% implies a less important shortage evidenced by mild to moderate bleeding. Although once considered fatal, factor replacement therapy using blood derivatives, FVIII or FIX concentrates, purified factors, etc. has proven to be an important advance in terms of life expectancy and quality of life. This serves as an example of the importance of genetic counseling, especially if we take into account that nowadays hemophilic males reach adulthood with minimal disabilities and often have children.
The sons of hemophilic fathers will neither have the disease nor pass it on to offspring.
All daughters of hemophilic fathers will be carriers and run a 50% chance of having carrier daughters or affected sons.
Sporadic cases, which occur approximately one-third of the time, can occur where a child born with hemophilia comes from a family that has no known history of bleeding disorders. It is estimated that the mother of a sporadic case has an 85-90% chance of being a carrier and initially a 42-45% chance of having affected sons or carrier daughters.
The mother is a carrier if she has more than one affected son.
If a hemophilic male marries a carrier female (a rare event unless they are blood-related), 50% of male offspring will be affected and the other 50% healthy, whereas 50% of female progeny will be affected and the remaining 50% carriers.
Women only have the disease if their father is hemophilic and their mother is a carrier (an exceptional case) or if there is sex-chromosome anomaly, as in the case of Turner Syndrome (45X).
Diagrams for points 1,2,3
The factor VIII gene is 186Kb in size and found on the q28 region of the X chromosome. Its large size contributes to a strong likelihood of there randomly existing numerous mutations or alterations in the gene, which explains the heterogeneity of the clinical expression of hemophilia A. 40-45% of severe cases are due to intron 22 inversion of the gene resulting from homologous recombination between the intragenic and extragenic copies. Less frequent are the deletions or point mutations (single base substitutions), which widely differ from one family to the next.
The factor IX gene is 34Kb in size and found nearby in the q26 region of the X chromosome. Different sized deletions and point mutations are the molecular mechanisms responsible for the clinical expression of hemophilia B. Unlike intron 22 inversion in hemophilia A, there is no principal mutation in hemophilia B, giving rise to its largely heterogeneous nature. For both types of hemophilia, identifying the mutation in one family member allows for a simple prediction about the rest of the family members. Each gene has coding parts called EXONS and non-coding parts for proteins called INTRONS, which are spliced out by RNA. A DNA analysis can be one of the following:
Direct, for when the mutation is known. Offers reliable diagnosis.
Indirect or linkage, for when it is impossible to identify the mutation. A highly reliable, but probabilistic diagnosis. The intragenic or extragenic markers utilized include RFLP (restriction fragment length polymorphisms obtained after exposing the gene to different enzymes) and VTNR or minisatellites (regions of the genome characterized by a number of tandem repeats that vary from one individual to another). Because the degree of informativity of each polymorphism varies, results are improved by combining several of them.
Until a few years ago, detecting female carrier status was done in analytic studies or using a statistical estimate of their genotype based on their family tree. Combining these techniques made it possible to predict 70-95% of potential carriers, although there was error with respect to individual detection. Two types of carriers can be classified using a family tree:
Obligate carriers, who are the daughters of hemophilic fathers or who have had more than one hemophilic son and
Potential carriers, who are the daughters of an obligate carrier.
Complementing pedigree analysis with analytical testing did not justify classifying a potential carrier as normal when their test results were normal.
Thanks to molecular biology, which allows us to determine specific mutations in each family, we can now identify with certainty the majority of carriers. However, it is still extremely important to identify potential female carriers using a family tree. Because diagnosis at the population level is often difficult, it is done starting at the individual level when, for example, hemophiliacs, adults or small children and their parents seek diagnostic testing. Before carriers decide to have children, it is very important to inform them of the risk of transmission.
Genetic counseling, although complex in certain circumstances, can be very useful when trying to correctly identify mutations, especially in the following cases: Sporadic cases: when trying to determine with certainty a mother’s carrier status, potential carriers and a mutation’s origin. Families with more than one hemophilic brother, but no other affected family members in previous or subsequent generations, which suggests evidence for germinal mosaicism (two cell lines, a healthy one and a mutated one, in gonads during germ cell development). When analyzing a hemophilic patient is impossible because they have died, analyzing the mother or another female at risk allows for identifying the defect causing the disorder.
Hematology (Dr. Uranga) and Immunology’s Molecular Biology Division (Dr. de Juan) are responsible for carrying out genetic analyses and making family trees. Educating and informing patients is carried out by Hematology (Dr. Uranga) with the help of The Hemophilia Association of Guipuzcoa.
My genetic-counseling services for hemophilia correspond to this section: Prepregnancy counseling and prenatal diagnosis. Couples at risk of having affected children are sent to us either because the wife is a carrier or because the husband has the disease.
Normally, these couples know exactly what the risks of transmission are, but our job is to confirm that risk and explain prenatal diagnostic methods used. It is very important to determine the carrier status of women who are potential carriers, bearing in mind the best case scenario occurs when prenatal diagnosis is not necessary because it has already been discarded that the woman is a carrier.
Prenatal diagnosis affords the opportunity to have healthy sons, restricting the option to abort for cases where the fetus is a hemophilic male. We must ask ourselves if prenatal diagnosis is worth it for those couples who do not want to terminate pregnancy and accept the risk of having affected sons. We do not believe it is, as a diagnostic test can be done on a newborn child (6 in every 10 hemophilic newborns experience perinatal bleeding).
Until before the application of molecular techniques, prenatal diagnosis could not be carried out until the 2nd trimester of pregnancy. After determining the sex of the fetus, which shows whether or not the fetus is at risk, a diagnostic test consisting of taking a fetal sample was done on male fetuses at least 17 weeks old. A fetal blood sample was taken by either fetoscopy or cordocentesis using advanced imaging ultrasound. The diagnosis, with a less than 1% error margin, was done using hematological, biochemical or immunological methods.
Current molecular biology not only provides excellent test results, but also allows for carrying out diagnostic tests during the 1st trimester. Immunologic and coagulation tests are now only used when DNA analysis is ambiguous or when pregnant women are well into their 2nd trimester.
Stages of prenatal diagnosis for hemophilia:
Genetic counseling: genetic and clinical information
Determining the sex of the fetus
CVS at 9-11 weeks (patient’s choice)
Amniocentesis at 15-16 weeks
Taking a fetal sample
CVS or Amniocentesis: DNA
Fetoscopy: fetal blood sample (19-21 weeks)
Cordocentesis: fetal blood sample (at least 17 weeks)
Diagnosing hemophilia from the fetal sample
DNA or molecular study: direct or indirect analysis
Fetal blood sample: determine levels of clotting factors VIII and IX, etc.
Quality controls must be used when taking fetal blood samples to assure there is no contamination from the mother. CVS (chorionic villus sampling) is currently the most preferred technique because it allows for determining the sex of the fetus, as well as doing a molecular analysis whose results are available in 3 to 7 days. Unlike amniocentesis, CVS permits earlier diagnostic testing (in the 1st trimester) and faster test results; the average waiting time for amniocentesis results is three weeks.
Consequently, if the fetus proves to be affected, we can avoid performing late abortions, and thus reduce the risk, patient complications and psychological repercussions associated with terminating a pregnancy.
We send the chorionic villi samplings to Dr. Tizzano of Hospital Sant Pau’s Genetic lab (Spain’s main geneticist and genetics center, respectively) for molecular analyses of hemophilia A and B. The ultrasound-guided CVS is carried out by Dr. Trecet of the Ultrasound Unit and is generally performed on women in their 10th or 11th week of pregnancy. The procedure can be performed via the vagina or via the abdomen depending on the location of the placenta, although we prefer the latter of the two options because we obtain better samples. The patient must sign an informed consent form explaining possible complications from the test and the risk of abortion, estimated to be between 2-3%.
Procedure: Transabdominally: we use the same 0.9 mm-wide needle used for amniocentesis and no anesthesia is necessary (see attached document).
Transcervically: Using ultrasound as a guide, a long flexible catheter less than 2 mm-wide is inserted through the cervix and into the uterus. It is not necessary to use anesthesia or dilate the cervix (see attached document).
In the last three years we have performed four prenatal diagnostic tests using CVS, all of which were successful and free of complications. There was only one male fetus, which turned out to be healthy.
Diagram of abdominal and vaginal transcervical procedures
I am a carrier, but I carry neither good news nor a bundle of provisions on my back. I am a carrier of hemophilia. Technically, I know one of my genes is mutated.
Emotionally, I regret that my children could inherit not only my height or eye color.
For a long time I’ve thought about whether I had the “right” to risk passing on this defect and if it was fair that my son “suffer” something that, despite having no physical signs of the disorder (and perhaps precisely for that reason), I have been suffering from the moment I realized I was a carrier. I even started to think that all science wanted me to do was question myself about something so essential for the majority of women as is the right to maternity. I wished then I hadn’t known about my carrier status, that way I could feel less responsible. I didn’t want my pregnancy to be a game of chance that meant waiting to see if I was lucky and it was the other chromosome’s “turn”. When you have a child, it’s not only about those “little blue eyes” and “kiss curls”. And less so about it being a “hemophiliac”.
It’s Oscar or Javier or Maria. It’s a PERSON, even if their eyes are brown and their blood takes a little longer to clot.
Treating carriers begins at the start of the diagnostic process so as to be able to plan for the future. All carriers need detailed information about what their condition means, what it implies and different options modern technology and resources can offer them.
Each person will need comprehensive and personalized social and psychological support, especially during pregnancy or prenatal diagnosis or when trying to find a partner, plan a family, etc. Furthermore, symptomatic carriers often have additional doubts and necessities.
A simple blood analysis can determine factor-activity levels in the plasma and, generally speaking, the severity of bleeding episodes in each patient.
The majority of hemophilic carriers fall within the normal range for factor-activity levels (50 – 150%) or are slightly below this range. However, these levels can vary a lot or even reach very low levels (10 –20% below the normal limits), which may cause excessive bleeding during menstrual periods, surgery, severe traumatisms, dental extractions or while giving birth. Depending on the levels of clotting factors VIII and IX, as well as the type of procedure to be performed, it may be necessary in the aforementioned circumstances to use the same products used by hemophiliacs (factor concentrates, desmopressin, etc.) prior to such procedures so as to avoid hemorrhaging. In cases of excessive menstrual bleeding, birth-control-related hormonal treatment may constitute an option when used with desmopressin and oral anti-fibrinolytics.
“Hemofilia: portadoras” Real Fundación Victoria Eugenia. Federación Española de Hemofilia. Servicio de publicaciones-número 00-2001. XXIII Congreso de la Federación Mundial de Hemofilia . La Haya, Países Bajos, 17-21 de mayo de 1998.