Hemophilia A hemophilia is an inherited disease caused by adeficiency or abnormal function of coagulation factors VIII.Hemophilia A is a genetic allele of recessive allele on chromosome
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1 The necessity of the project
Along with the development of the economy and the society, theinfertility rate is increasing and is a concern for many couples Born inthe 1970s of the last century, it can be said that in vitro fertilization "invitro fertilization - IVF" has become a lifeline for many infertile coupleswho want to have children This method has brought joy and happiness
to many couples all over the world, but not every couple is lucky to havethe desired children by this method The success rate of this method isonly more than 35% depending on the country There are many factorsaffecting the success of this method such as age of parents, health status,psychological spirit, quality of sperm and eggs and especially quality
of embryos after fertilization Embryos that may be abnormalchromosomes (chromosomes) or sex chromosomes all reduce thepossibility of pregnancy after implantation of the embryo into the uterus,which may cause miscarriage or birth of unwell children strong Toreduce the rate of birth of unhealthy children and increase the success rate
of in vitro fertilization methods, scientists have recently applied embryoscreening process "Pre-implantation Genetic Diagnosis, PGD "is alsocalled" genetic diagnosis before nesting "to get the best embryos forembryo transfer
Hemophilia A (hemophilia) is an inherited disease caused by adeficiency or abnormal function of coagulation factors VIII.Hemophilia A is a genetic allele of recessive allele on chromosome Xwithout alleles on the Y chromosome, so the mother carrying thedisease gene will inherit the son and clinical manifestations According
to the statistics of the world hemophilia A, currently there are about250,000 patients with hemophilia A and only about 50,000 are treatedspecifically In Vietnam, an estimated 6000 people have hemophilia Adisease and about 30,000 people carry the hemophilia disease gene Themain clinical manifestation is bleeding anywhere in the body, andprolonged bleeding: articular bleeding, internal bleeding, cerebralbleeding, bleeding in the neck and chest Clinical manifestations isdirectly related to the concentration of factor VIII in plasma
Trang 2The screening of prenatal hemophilia A is very important to help thebirth of perfectly healthy babies The identification of mutations ofpathogenic genes, screening of healthy people carrying disease genes andprenatal diagnosis of hemophilia A have been studied quite a lot inVietnam The management of patients and carriers of this disease is apremise firm to successfully implement the pre-implantation diagnosisprocess in embryo screening This method will help reduce complicationsand sequelae for mothers during pregnancy, reduce pregnancytermination by detecting fetal disease when diagnosing prenatal, bringinghappiness to couples carrying disease gene, while reducing the rate ofdisease in the community Along with the development of molecularbiology, many techniques have been applied to detect hemophilia Amutations such as multiplex polymerase chain reaction (PCR), localfluorescent hybrid technique (FISH) The microsatellite DNA technique
is a technique that uses fluorescence-binding primers of short tandemrepeat (STR) marker and analyzes their size through capillaryelectrophoresis on a gene sequencing machine to determine chromatinanomalies can This technique has many advantages due to its highsustainability, easily detecting heterozygotes without identifying directmutations, thus reducing the cost of the patient's family Currently, thistechnique is quite new in Vietnam for the application of diagnosis ofgenetic diseases
2 The objectives of the Project
1 Determination of healthy people carrying hemophilia A gene by microsatellite DNA technique.
2 Pre-implantation diagnosis for mothers with hemophilia A gene
by microsatellite DNA technique.
3 Scientific and practical significance of the topic
Genetic diseases are a group of diseases, although not as common assome other groups of diseases, but in fact, every year there are largenumbers of babies born with malformations or genetic diseases.Hemophilia A is a disease that is difficult to treat and has very seriousconsequences on health, spirit and quality of life Therefore, the problem
is to resolve the earliest to reduce the rate of sick children Hemophilia Adisease has been passed down through generations and many people get
Trang 3sick in the same family Identifying genetic mutations and detectinghealthy people carrying disease genes is necessary to have appropriateadvice to help prevent and reduce the rate get sick Antenatal diagnosis is
a common measure implemented in Vietnam for general genetic diseases,hemophilia A disease in particular helps mothers have the ability toproduce healthy, technically cheap children, although However, a healthychildbirth also depends on the random probability of pregnancy, in casethe unborn child must suspend pregnancy at greater than 17 weeks ofgestation thus affecting the physiology and health of the mother Pre-implantation diagnosis will help actively select uninfected embryos toimplant the uterus This study used Dna microsatellite technique in pre-implantation diagnosis of hemophilia A This is an indirect technique todetect heterozygous mutant alleles without identifying direct mutations
4 The thesis structure
- The thesis is presented in 139 pages (excluding references andappendices) The thesis is divided into 6 parts:
+ Introduction: 3 pages
+ Chapter 1: Overview: 42pages
+ Chapter 2: Research subjects and methods: 18 pages
+ Chapter 3: Research results: 45 pages
+ Chapter 4: Discussion: 30 pages
+ Conclusion: 1 page
The thesis consists of 4 tables, 35 figures Use 107 references including
Vietnamese and English The appendix includes: Amino acid encoding,Summary table using 4 markers to amplify 112 members (patient +
of the family hemophilia A
Trang 4Chapter 1 OVERVIEW 1.1 GENERAL CHARACTERISTICS OF HEMOPHILIA A 1.1.1 Definition
Hemophilia A disease is an inherited disease due to deficiency orabnormal function of blood clotting factors - deficiency of factor VIIIcauses hemophilia A, factor IX deficiency causes hemophilia B,deficiency factor XI causes hemophilia C Hemophilia A disease is themost common genetic hereditary disorder
1.1.2 Diagnosis of disease
Defined diagnosis
- Based on clinical: There are bruises, hematoma, bleeding
- Based on family history
- Based on blood tests:
+ Normal platelet count, PT (Prothrombin Time: normalprothrombin time), normal fibrinogen, APTT (Activated PartialThromboplastin Time: partially activated thromboplastin time) lasts>1.5 normal values
+ The activity of plasma VIII factor decreases below 40%
+ Von Willebrand factor normal
+ Other tests: Mixtest determines antibodies to factor VIII
Diagnosis of disease level
Level
Concentration of factor VIII (%) (active (UI / ml))
Natural bleeding is not related
to injury, often bleeding injoints and muscles
Trang 5Gene-carrying
father
Normal daughter Gene-carrying
daughter Normal son Sick son
Normal mother
Gene-carrying daughter Normal
son Sick father
1.2 GENETIC CHARACTERISTICS AND MOLECULAR BASIS OF HEMOPHILIA A
1.2.1 The genetic mechanism of hemophilia A
This is an inherited disease associated with gender: In men there isonly one X chromosome so if the X chromosome carries the disease, theamount of factor VIII is not enough and the person suffers fromhemophilia A For women, thanks to two X chromosomes, if an Xchromosome carries the disease, the normal gene on the otherchromosome still synthesizes normal factor VIII The woman does notget sick but carries a diseased gene that will pass on the son and the sonwill get sick, if passed on to the daughter, the girl will become thecarrier of the disease
Figure 1.1: Genetic diagram of the mother carrying the married gene to
normal father
Figure 1.2: Genetic diagram of a normal mother getting married to a sick father
Trang 61.2.2 Molecular basis for studying hemophilia A disease
Since 1984, research by Vehar and colleagues showed a fullunderstanding of the molecular structure of the F8 gene that synthesizesfactor VIII protein, paving the way for studies on the molecularmechanism of Hemophilia A and its types F8 gene mutations causedisease
In the endogenous coagulation pathway, factor VIII acts as acofactor with activated factor IX (IXa) in the presence of Ca2 + andplatelet phospholipid activating the X-Xa factor, thereby affecting theformation of thrombin from prothrombin, helping to create and fixfibrin from fibrinogen, perfecting the process of clot formation withplatelets and vascular elements The deficiency or abnormal functionfactor VIII stops endogenous cascade cascades following endogenouspathways, leading to prolonged, uncontrolled bleeding in hemophilia Apatients
1.3 HEALTHY PEOPLE CARRYING THE GENE OF HEMOPHILIA A
Hemophilia A is a genetic disorder caused by recessive geneslocated on sex chromosome X, caused by factor VIII mutation Thediagnosis of healthy people carrying disease genes is very important toprevent the birth of children with hemophilia A
1.3.1 General characteristics of people carrying disease genes
A healthy person with a disease gene is a person with an Xchromosome with an F8 mutation and a normal X chromosome.Clinical manifestations may be severe or mild
So women definitely carry disease genes when they have one of thefollowing conditions: Have a father with hemophilia A disease; At leasttwo sons have hemophilia A; Have a son who is ill or has a sentence or
a younger brother who is ill or a man who is related by blood tohemophilia A; There is a sick son and there is certainly a woman in thefamily who carries the disease gene
Women are likely to carry disease genes when at least one person inthe maternal family is ill but does not have a sick child, or has a sonwith Hemophilia A without any other member being ill
Trang 71.4 IN VITRO FERTILIZATION METHOD (IVF) AND IMPLANTATION DIAGNOSIS PROCEDURE (PGD)
PRE-1.4.1 In vitro fertilization method (IVF)
The success of in vitro fertilization was marked by the birth of thefirst child under this method in 1978 On average in countries likeSweden, Australia and the United States, about one in every 50-80babies is born with in vitro fertilization Every year, more than 120,000couples in the United States perform in vitro fertilization In the future,the number of children born with this method will be increased by theincrease in the average age at marriage, the average age at firstpregnancy and the increase in the rate of infertility among couples.husband in the world
1.4.2 Pre-implantation genetic diagnosis technique (PGD)
PGD technology is now a complete technique, allowing couples atrisk of transmitting genetic diseases to children who may have theopportunity to have a baby without the diagnosis of prenatal care afterpregnancy and abort the fetus when detecting pregnancy PGDtechnique is based on the detection of embryonic genetic abnormalities(in-vitro) and only re-implants into the uterus of embryos withoutgenetic abnormalities
1.4.2.3 Indications of pre-implantation diagnosis
Today PGD is applied to diagnose about 170 different pathologies.Increasingly, scientists have identified more genes related to pathologiesand identified the types of pathogenic mutations of these genes Thisallows PGD to diagnose the embryos without disease genes to select andimplant these embryos into the uterus Subjects when in vitro
fertilization are encouraged to use this procedure including: i) Couples who carry genes that cause genetic diseases on chromosomes are often
or associated with sex chromosomes ii) People with chromosomal anomalies such as segment loss, reversal, repeating and segment transitions iii) Older women, women exposed to chemicals, radiation
to avoid possible and genetic chromosomal abnormalities for their children iv) Women with a history of miscarriage and multiple pregnancies v) Couples who have previously performed the IVF method but have been unsuccessful and have no known cause.
Trang 81.4.3 The latest molecular biology methods and techniques apply
in pre-implantation diagnosis
1.4.3.1 PCR and Multiplex-PCR
PCR is the first technique applied to PGD by Handyside et al In
1990 when identifying repeated sequences on Y chromosomes todetermine the sex of embryos in the diagnosis of genetic diseasesassociated with sex chromosomes and transfer uninfected embryos intothe uterus
1.4.3.2 Fluorescence in situ hybridization (FISH)
On-site fluorescent hybrid technique (FISH) is a molecular-cellgenetic technique that uses fluorescent-based DNA transducers totarget specific DNA sequences on the chromosomes of cells ininterstitial or infectious chromosomes in the middle period, therebydetecting the presence or absence of a certain gene segment byfluorescence microscopy
1.4.3.3 Microsatellite DNA
Microsatellite DNA was developed based on standard PCRtechniques with the use of fluorescent-mounted primers and anautomated DNA sequencer to identify PCR products With specificfluorescence probes coupled with computer-enhanced imagingtechniques, amplified fluorescent DNA fragments can be detected at amuch lower concentration than the determination of DNA on the gel.electrophoresis agarose or acrylamide This technique has high accuracyand reliability even when performing a reaction to identify genescarrying mutations in heterozygotes In recent years, this technique hasbeen further developed using polymorphic markers on chromosomes todetermine the presence of different alleles This technique is based onpolymorphic information of short repeat sequences (short tandem repeat:STRs)
Trang 9Chapter 2 RESEARCH SUBJECTS AND METHODS
2.1 Research subjects: 35 families of hemophilia A patient:
- 35 hemophilia A patients were identified with genetic mutations atthe Center for Genetic-Protein Research, Hanoi Medical University
* Diagnostic criteria for hemopphilia A:
+ Clinical:
- Bleeding: Difficult to stop bleeding after injury, collision or naturalbleeding
- Location: Bleeding in joints, muscles or some other positions
- Nature: Often recurrent bleeding
- Prehistoric history: a history of prolonged bleeding or in a familywhere a loved one has bleeding that is difficult to hold
+ Subclinical:
- APTT lasts
- Quantitative FVIII, IX decrease below 30%
- Time for blood to flow normally
- Normal platelet count and platelet concentration
- Normal Prothrombin
- Normal von Willebrand factor
* Exclusion criteria:
+ Patients with Von-Willebrand disease
+ Genetic diseases that cause prolonged APTT: reduction of factor XI,XII, prekallikrelin
+ Circulating disease resistant to FVIII: autoimmune disease (lupus)
2.2 Research Methods
Cross-sectional descriptive descriptive study
Trang 102.3 Time and site of the study
+ Center for Gen-Protein Research, Hanoi Medical University.+ Time from 12/2015 - 5/2018
2.4 Ethics in the research
The topic has been approved by the Ethics Council of Hanoi MedicalUniversity under the Decision No.187/HDĐĐHYHN, dated February 20,2016
2.5 Funding for the project
The project is carried out with the support of the funding of the
state branch project “Building pre-implantation diagnosis procedure
by microsatellte DNA technique to screen some genetic chromosomal diseases of sex chromosomes” under the program KC04.17/11-15.
2.6 Process and techniques used in the study
The techniques used in the study include: Collecting patientinformation according to research medical records DNA extractiontechnique from peripheral blood samples Technique of sequencing toidentify gene mutations, process of detecting healthy people carryingdisease genes by Dna microsatellite technique, Process of pre-implantation diagnosis of hemophilia A by microsatellite DNAtechnique The research process follows the diagram
RESEARCH DIAGRAM
Trang 11* Designing the research on families treated with PGD
Chapter 3 RESEARCH RESULTS 3.1 RESULTS OF DETECTING HEALTHY PEOPLE CARRYING THE MUTATED F8 GENE WITH THE MICROSATELLITE DNA TECHNIQUE
3.1.1 Results of identification of heterozygous markers of F8 gene
in patients and carriers of disease genes by microsatellite DNA technique
Chart 3.1: Results of homozygous and homozygous results of STR
Comments: The results showed that with the use of 4 FXS
Trang 12markers 1108, DXS 9897, F8int22, DXS9901, the two markers DXS9897 and DXS9901 gave a heterozygous rate of 55.2% higher Therefore, the research will prioritize using this marker first.
3.1.2 Results of detection of healthy people carrying F8 gene were mutated by microsatellite DNA technique
The results compare the two methods to identify healthy peoplecarrying the disease gene hemophilia A
Table 3.1 Compare the results of detection of healthy people carrying
disease genes by two methods
Patient Exon Mutant form member Family
Sequencing Microsatellite DNA Gene
carryi ng
No gene carryi ng
Gene carry ing
No gene carryi ng
HA16 14 c.4996-4997insAThr1904Asn fs*2
Comments: The results showed that the detection of healthy
people carrying disease genes by genome sequencing andmicrosatellite-DNA techniques completely coincided
Trang 13Figure 3.1 Image of genome sequence of family code HA16 (Red
arrow indicates mutant position) Comments: Exon sequencing images of 14 F8 genes of mother and
uncle of HA16 patients appeared overlapping vertices after themutation point c.4996-4997insA, indicating that the mother and uncle
of the patient had mutated F8 gene in the state heterozygous Thepatient's aunt does not appear mutations, so her aunt is a normalperson