Genetics of Hemophilia

Hemophilia is a hereditary bleeding disorder affecting predominantly males, with an incidence of 1 in 10,000 to 1 in 50,000 boys. This disease is caused by a defect, or change in DNA (mutation), in a single gene. In the case of hemophilia A, it is the gene for FVIII; in hemophilia B, it is FIX. Hemophilia has so-called X-linked inheritance, because both genes are located on the X sex chromosome, which causes the different incidence of this disease in women and men. These characteristics allow for genetic diagnostics of hemophilia, which involves identifying mutations in affected individuals and detecting carriers in these families, among other activities carried out by the Laboratory for Hemostasis Disorders.

Carrier detection was already being performed in the 19th century based on pedigree analysis. For example, if a sister of a man affected by hemophilia had a son who was also affected by hemophilia, it can be said that this woman is a carrier. Likewise, all daughters of an affected man are carriers. In the Czech Republic, 55% of cases involve a family history of hemophilia (familial form), while the remaining 45% are cases without prior family occurrence (sporadic form). In 1981, carrier detection using biochemical analysis was introduced; however, this method was not applicable to all cases. From 1988, genetic methods based on DNA analysis began to be used. First the RFLP method (Restriction Fragment Length Polymorphisms), then DNA sequencing. Genetic analysis can be divided into two types: linkage analysis (indirect diagnostics) and mutation identification (direct diagnostics). Simply put, linkage analysis searches in the family of the affected individual for a genetic marker that would be inherited together with the segment containing the mutation. The advantage of this diagnostics is that it is not necessary to know exactly where the mutation occurred. However, DNA from the affected individual and as many relatives as possible must be available. It can also happen that the analysis performed will not be useful and will not determine whether the person examined is a carrier. Linkage analysis cannot be used if the mother is not a carrier. In other words, if it is a new mutation. In the Czech Republic, 13% of mothers of hemophilia patients are not carriers. In such cases, direct diagnostics is performed, which involves identifying the exact segment of the gene where the mutation occurred. This diagnostics is very accurate but time-consuming and costly, because almost every family has its own mutation.

Dr. Hrachovinova stated that in the Czech Republic there are currently 875 patients with hemophilia A (according to data from the national registry of congenital bleeding disorders), of whom a mutation leading to the condition has been found in 441. Overall, mutation screening was performed on 641 patients with hemophilia A. In the case of patients with hemophilia B, a mutation was identified in 73 out of 74 analyzed patients. Summary data from 1989-2011 show that 602 carriers of hemophilia A were identified from a total of 399 families, and 96 carriers of hemophilia B from a total of 180 families.

The Laboratory for Hemostasis Disorders closely cooperates with medical genetics centers in Prague, Brno, Ostrava, Olomouc, and other cities, where it provides genetic and hematological consultations. According to prenatal diagnostics data (i.e., diagnostics of the child before birth) from 1992-2011, out of a total of 188 analyses performed, 42 male fetuses with hemophilia A and 3 male fetuses with hemophilia B were identified. The Gennet Clinic currently performed preimplantation genetic diagnostics (diagnostics of the embryo before its implantation into the uterus of the future mother) for four families, resulting in the birth of three children. This diagnostics involves taking one cell from the embryo on day 3 or 5 after in vitro fertilization for genetic analysis. In conclusion, Dr. Hrachovinova stated that she is currently intensively cooperating with IVF centers to increase options for carriers planning pregnancy.

The second lecture was titled "Prenatal Diagnostics and Preimplantation Genetic Diagnostics" and was authored by M. D. Flora Peyvandi, Ph. D., a researcher at the Center for Hemophilia and Thrombosis, University of Milan. Dr. Peyvandi spoke about prenatal diagnostics, both invasive and non-invasive. The lecture was also devoted to preimplantation diagnostics and the possibilities of its use in the future for diagnosing hemophilia.

Prenatal diagnostics represents a set of methods that can be performed during pregnancy and provide information about fetal development. These methods can be divided into invasive and non-invasive. Invasive methods include amniotic fluid sampling (amniocentesis) and chorionic villus sampling (choriocentesis). Non-invasive methods include ultrasound examination, analysis of fetal cells in maternal blood, and analysis of so-called "free" or freely circulating fetal DNA (deoxyribonucleic acid) in maternal blood. In the case of hemophilia patients, the mutation is first searched for, which can subsequently help in prenatal testing of their sisters or daughters who are carriers. A woman who is a carrier can undergo one of the invasive methods, or a non-invasive method (determination of fetal sex from maternal blood), or preimplantation diagnostics. The goal of all these methods is to distinguish an affected fetus from a healthy one; in the case of the non-invasive method, the decision is made solely based on the sex of the fetus.

Chorionic villus sampling, which involves finger-like projections of the placenta, can be performed at 10-12 weeks of pregnancy and is associated with approximately a 1% risk of spontaneous miscarriage. Amniotic fluid sampling is performed later (weeks 15-16) and carries a 0.5-1% risk of spontaneous miscarriage.

The non-invasive method of "free" DNA analysis was a breakthrough discovery in the field of prenatal diagnostics. This DNA originates from placental cells, is detectable in maternal blood from the 4th week of pregnancy, half of the molecules are degraded in maternal blood within 16 minutes, and no free fetal DNA is detectable 2 hours after delivery. "Free" fetal DNA provides a fetal sample for prenatal diagnostics without the risk of spontaneous miscarriage and the feelings of anxiety experienced by the mother during invasive prenatal diagnostics. Currently, "free" DNA is used for determining fetal sex, which is performed by analyzing segments on the Y chromosome (this chromosome is present only in males). The sensitivity of this method (the percentage of correctly identified male fetuses) is 73% at 4-7 weeks of pregnancy; however, if blood sampling is performed at 8 weeks or later, the sensitivity is 94%. Unfortunately, the analysis of "free" DNA currently has significant limitations: it is not possible to separate maternal DNA from fetal DNA. Sex determination is possible based on analysis of segments that the mother does not have. The damaged gene causing hemophilia is passed from the mother to her son, so it is not possible to distinguish whether the damaged gene found comes from the mother or the child. Therefore, methods are being developed to reliably separate fetal cells from maternal blood. These include, for example, the creation of a biochip or the separation of fetal cells using magnetic beads. Dr. Peyvandi stated that, according to the results of a survey by the British Society for Human Genetics, non-invasive fetal sex determination reduced the number of invasive prenatal diagnostics by 45% in women who had an increased risk of having a child with an X-linked disease.

An alternative to the above-mentioned prenatal diagnostics methods is preimplantation genetic diagnostics. This diagnostics is indicated in cases of hereditary diseases in the family, frequent miscarriages with suspected chromosomal abnormalities, and in couples who have chromosomal abnormalities preventing conception. In the case of hemophilia, there are two possible strategies. The first strategy is the selection of female embryos only, which means losing all male embryos, even though approximately half of them will be healthy. This diagnostics was first performed in 2004 in France. The second strategy is mutation identification and selection of embryos that do not carry the mutation regardless of sex. Mutation identification in preimplantation diagnostics was first performed for hemophilia in 2006 in the United Kingdom. Although preimplantation diagnostics looks very promising, it still has limitations: high cost, couples must undergo procedures associated with in vitro fertilization (in a test tube), diagnosis from 1-2 cells, and the associated risk of misdiagnosis. It can also happen that all embryos will be affected or will carry the mutation.

In conclusion, Dr. Peyvandi noted that advances in molecular technologies have increased the possibilities of non-invasive prenatal diagnostics and preimplantation diagnostics, which can now be considered a well-established method.

Anastassiya Zidkova anastazie.d@gmail.com