Phát hiện người lành mang gen bệnh và chẩn đoán trước làm tổ bệnh hemophilia a bằng kỹ thuật microsatellite DNA tt tiếng anh

1 INTRODUCTION The necessity of the project Along with the development of the economy and the society, the infertility rate is increasing and is a concern for many couples Born in the 1970s of the last century, it can be said that in vitro fertilization "in vitro fertilization - IVF" has become a lifeline for many infertile couples who 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 have the desired children by this method The success rate of this method is only more than 35% depending on the country There are many factors affecting 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 abnormal chromosomes (chromosomes) or sex chromosomes all reduce the possibility of pregnancy after implantation of the embryo into the uterus, which may cause miscarriage or birth of unwell children strong To reduce the rate of birth of unhealthy children and increase the success rate of in vitro fertilization methods, scientists have recently applied embryo screening process "Pre-implantation Genetic Diagnosis, PGD "is also called" genetic diagnosis before nesting "to get the best embryos for embryo transfer Hemophilia A (hemophilia) is an inherited disease caused by a deficiency or abnormal function of coagulation factors VIII Hemophilia A is a genetic allele of recessive allele on chromosome X without alleles on the Y chromosome, so the mother carrying the disease gene will inherit the son and clinical manifestations According to the statistics of the world hemophilia A, currently there are about 250,000 patients with hemophilia A and only about 50,000 are treated specifically In Vietnam, an estimated 6000 people have hemophilia A disease and about 30,000 people carry the hemophilia disease gene The main clinical manifestation is bleeding anywhere in the body, and prolonged bleeding: articular bleeding, internal bleeding, cerebral bleeding, bleeding in the neck and chest Clinical manifestations is directly related to the concentration of factor VIII in plasma The screening of prenatal hemophilia A is very important to help the birth of perfectly healthy babies The identification of mutations of pathogenic genes, screening of healthy people carrying disease genes and prenatal diagnosis of hemophilia A have been studied quite a lot in Vietnam The management of patients and carriers of this disease is a premise firm to successfully implement the pre-implantation diagnosis process in embryo screening This method will help reduce complications and sequelae for mothers during pregnancy, reduce pregnancy termination by detecting fetal disease when diagnosing prenatal, bringing happiness to couples carrying disease gene, while reducing the rate of disease in the community Along with the development of molecular biology, many techniques have been applied to detect hemophilia A mutations such as multiplex polymerase chain reaction (PCR), local fluorescent hybrid technique (FISH) The microsatellite DNA technique is a technique that uses fluorescence-binding primers of short tandem repeat (STR) marker and analyzes their size through capillary electrophoresis on a gene sequencing machine to determine chromatin anomalies can This technique has many advantages due to its high sustainability, easily detecting heterozygotes without identifying direct mutations, thus reducing the cost of the patient's family Currently, this technique is quite new in Vietnam for the application of diagnosis of genetic diseases The objectives of the Project Determination of healthy people carrying hemophilia A gene by microsatellite DNA technique Pre-implantation diagnosis for mothers with hemophilia A gene by microsatellite DNA technique Scientific and practical significance of the topic Genetic diseases are a group of diseases, although not as common as some other groups of diseases, but in fact, every year there are large numbers of babies born with malformations or genetic diseases Hemophilia A is a disease that is difficult to treat and has very serious consequences on health, spirit and quality of life Therefore, the problem is to resolve the earliest to reduce the rate of sick children Hemophilia A disease has been passed down through generations and many people get sick in the same family Identifying genetic mutations and detecting healthy people carrying disease genes is necessary to have appropriate advice 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 to produce healthy, technically cheap children, although However, a healthy childbirth also depends on the random probability of pregnancy, in case the unborn child must suspend pregnancy at greater than 17 weeks of gestation thus affecting the physiology and health of the mother Preimplantation diagnosis will help actively select uninfected embryos to implant the uterus This study used Dna microsatellite technique in preimplantation diagnosis of hemophilia A This is an indirect technique to detect heterozygous mutant alleles without identifying direct mutations The thesis structure - The thesis is presented in 139 pages (excluding references and appendices) The thesis is divided into parts: + Introduction: 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: page The thesis consists of tables, 35 figures Use 107 references including Vietnamese and English The appendix includes: Amino acid encoding, Summary table using markers to amplify 112 members (patient + patient's family) and a list of 35 patients hemophilia A and 77 members of the family hemophilia A Chapter OVERVIEW 1.1 GENERAL CHARACTERISTICS OF HEMOPHILIA A 1.1.1 Definition Hemophilia A disease is an inherited disease due to deficiency or abnormal function of blood clotting factors - deficiency of factor VIII causes hemophilia A, factor IX deficiency causes hemophilia B, deficiency factor XI causes hemophilia C Hemophilia A disease is the most 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: normal prothrombin time), normal fibrinogen, APTT (Activated Partial Thromboplastin 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 Concentration of Level factor VIII (%) Symptoms of bleeding (active (UI / ml)) Serious Natural bleeding is not related (Accounting C p.lle1698 Thr HA50 14 c 4026G>T p Glu1342stop HA61 Family member Mother Aunt Aunt Mother Sister Amniotic fluid Mother Sister Mother Uncle Cousin Cousin c 435450del15bp) Mother p.135 -139 Tyr-Val + + - + + + + + + - + + + + + + + + - - + + + + Comments: The results showed that the detection of healthy people carrying disease genes by genome sequencing and microsatellite-DNA techniques completely coincided 13 Figure 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 the mutation point c.4996-4997insA, indicating that the mother and uncle of the patient had mutated F8 gene in the state heterozygous The patient's aunt does not appear mutations, so her aunt is a normal person 14 Figure 3.2 Repeated sequence electrophoresis images use HA16 family DXS9901 and DXS989 primer pairs Comments: The above results show that in the sequence of repeating DXS 9798, DXS9901, the patient's mother appeared vertices, each vertex corresponds to allele At the peak coincides with the peak of the patient is the peak of the mutant allele , ie the patient will receive this mutant allele from the mother, the mother of the patient carries the mutant F8 gene in heterozygous state The patient had two peaks coinciding with the mutant allele of the patient, so he had the disease gene The patient's aunt has two peaks that not match the patient, so the aunt is a normal person Results of identification of healthy people carrying disease genes by microsatellite DNA technique Table 3.3 Rate of detection of healthy people carrying disease gene by microsatellite-DNA technique 15 Gene No gene Subcarrying carrying total n (%) n (%) The mother of the patient 35(100) 0(0) 35 Other female members 33(78,6) (21,4) 42 Total 68/77(88,3) 9/77 (11,7) 77 Notes: Other female members include: sister, sister, aunt, aunt, grandmother, cousin, patient's cousin 3.2 RESULTS OF PRE-IMPLANTATION DIAGNOSIS IN HEMOPHILIA PATIENTS WITH MICROSATTELITE DNA TECHNIQUE Family member Figure 3.3 The multiplex PCR results identified HA28 family normal male embryos Comments: The above results show that there are peaks in the marker area of Amel corresponding to the sex chromosome X and Y, and peak in the sex marker area SRY corresponding to the sex chromosome Y shows that this is the embryo male With FXS1108 marker, the embryo appears peak size 166bp corresponding to the peak of normal allele, no peak size of 177bp corresponds to the peak of the mutant allele Similarly, with the embryo DXS9897 marker also appears a peak of 260 bp corresponding to the peak of normal allele, does not appear peak size of 256bp corresponding to the peak of the mutant allele This result confirms that this is a normal male embryo without the mutant F8 gene of the mother 16 Figure 3.4 The multiplex PCR result identified the male HA28 family pathological embryo Comments: The results show that there is a peak in the sex marker area of SRY corresponding to Y sex chromosome, showing that this is a male embryo With the FXS 1108 marker, the embryo appears with a peak size of 177bp corresponding to the peak of the pathological allele coinciding with the patient's allele peak, not the peak size 166bp corresponding to the peak of the normal allele Similarly, with the embryo DXS9897 marker, there is also a peak of 256 bp corresponding to the peak of the pathological allele coinciding with the patient's allele peak, not the peak size of 260 bp corresponding to the peak of normal allele With the above results, it can be confirmed that this is a pathological male embryo with mutant F8 gene of the mother 17 Figure 3.5 The PCR multiplex result identified female embryos carrying the HA28 family gene Comments: The results show that there is a 103 bp peak in the marker area Amel X, which is an X chromosome marker, having the same sequence of regions between the two X chromosomes, so there is only one peak This result proves that this is a female embryo With DXS FXS1108 marker, the embryo appears 177bp size corresponding to the peak of the mutant allele (pathological allele) Similarly, with the DXS9897 marker in embryos, peaks of 256 bp size correspond to the peak of the mutant allele Both markers not appear as normal peaks This result can confirm that this is a female embryo carrying the gene 18 Figure 3.6 The PCR multiplex result identifies female embryos without the HA28 family gene Comments: The results show that there is a 103 bp peak in the marker area Amel X, which is an X chromosome marker, having the same sequence of regions between the two X chromosomes, so there is only one peak This result proves that this is a female embryo With DXS1108 marker, the embryo appears 166bp in size corresponding to the peak of the normal allele Similarly, with the DXS9897 marker in embryos, the peak size of 260 bp corresponds to the peak of the normal allele Both markers not appear the peak of the pathological allele This result can confirm that this is a female embryo without the gene 19 * HA28 embryo genealogy diagram Figure 3.7 HA28 family genealogy diagram Comments: HA28 family after in vitro fertilization, biopsy stopped diagnosing 01 normal male embryo (II2), normal female embryo (II5), 01 abnormal male embryo (II3), female embryo carrying disease gene (II4), 20 * Embryo ratio after gene diagnosis by microsatellite DNA technique 34.70% 65.30% Phôi không bất thường gen Số phôi bất thường gen Chart 3.2: Rate of abnormal and non-abnormal embryos in genes Comments: Using microsatelite DNA technique in pre-implantation diagnosis of 12 families Hemophilia A patients have a wish to have a healthy child Of the 75 embryos that were biopsy for genetic testing, 26/75 embryos detected mutations of the gene causing hemophilia A, including male embryos and female embryos, accounting for 34.7% The remaining 49/75 embryos, accounting for 65.3%, have normal phenotype; Including normal male and female embryos and female embryos with genes Chapter DISCUSSION * Results of identifying healthy people with disease genes by microsatellite DNA technique 21 Hemophilia A disease is hereditary due to deficiency or abnormal function of factor VIII, with a ratio of 1-2 in 10,000 worldwide, there is no difference in race [85] Since 1984, from the study of Gitschite and colleagues [86] has shown a full understanding of the molecular structure of F8 gene synthesizing factor VIII protein: F8 gene is one of the major genes of the body, with 186 kb size including 26 exon FVIII factor is a large glycoprotein that acts as a co-factor necessary for the activation of proteolytic activation of factor X activated by factor IX in the endogenous pathway The F8 gene encodes for FVIII protein consisting of 2332 amino acids and has a molecular weight of about 300 kD When the F8 gene was mutated, the cell decreased or lost the ability to synthesize FVIII factor causing hemophilia A A full understanding of the molecular structure of F8 paved the way for studies on the mechanism of molecular hemophilia A and F8 gene mutations cause hemophilia A disease The birth and development of molecular biology helps detect mutations and mutations in the F8 gene causing hemophilia A Currently, there are many new mutations published annually on the database HAMSTeRS (Hemophilia A Mutation, Search, Test and Resource Site) is the information management site for British hemophilia A: by January 2013, there were 2158 mutations in different positions on the published F8 gene or on the CDC database (The Centers for Disease Control and Prevention) is the US information management site for hemophilia A, a total of 2556 disease-causing mutations have been published Currently, many different methods have been used in the world to detect healthy people carrying hemophilia A In 2004, Le Nhat Minh and colleagues studied healthy people carrying hemophilia A gene using cutting enzyme technique limit (PCR-RFLP) In 2014, Bui Thi Thu Huong and colleagues used I-PCR techniques and gene sequencing techniques to identify healthy people carrying disease genes in families who identified point mutations on patients Currently, the detection of healthy people carrying disease genes is usually done by the technique of detecting direct mutations in the F8 gene based on the patient's mutation However, the cost of detecting F8 mutations is quite high due to its large size (186kb) with 26 exon [85] and not all patients have 22 genetic testing conditions, among others Human genes were tested, there was still a certain percentage (about more than 10%) of patients who had not detected F8 gene mutations Stemming from that fact our team performed indirect microsatelliteDNA method to identify healthy people carrying the disease gene hemophilia A on female members of the patient family hemophilia A unidentified mutation points to Identify heterozygous markers and have genetic counseling and pre-implantation diagnosis for families who need a perfectly healthy baby The study carried out on 112 members of hemophilia A families with the use of STR obtained the heterozygous rate at STR DXS 9897 and DXS 9901 was the highest of 55.2%, while STR F8in22 and FXS1108 heterozygous lower death The more that the higher the polymorphism, the higher the heterozygous rate Therefore, with the use of high polymorphic STR, the ability to detect healthy people carrying higher disease genes is significant in managing hemophilia A patients in particular and genetic diseases in general Thus, the detection of high polymorphism of STR will be applied in detecting healthy people carrying genes and pre-implantation diagnosis of genetic diseases Thus, when simultaneously performing two techniques of sequencing and microsatellite-DNA on members of families, patients HA61, HA16, HA37, HA30, HA50 have identified the point mutation, we also obtained the same results There are 13 female members (mother, grandmother, aunt, and patient) who carry disease genes and female members (amniotic fluid of patients' mothers, cousins) who not carry disease genes Thus, the microsatellite-DNA process uses four fluorescently labeled primers DXS9897, DXS9901, FXS1108, DXS9901, and F8int22 to amplify repeating sequence regions STR standardized multiplex PCR process, PCR amplification products were electrophoresis on the Beckman coulter system and analyzed the results with the Genome lab system software as we built to identify healthy people carrying the correct hemophilia A gene and to give accurate results gene sequencing method Therefore, the microsatellite-DNA technique has been standardized to apply in pre-implantation diagnosis With microsatellite DNA technology has many advantages compared to other techniques because of its higher 23 sustainability, requiring less technical manipulation, suitable for diagnosis of large sample size, pre-implantation diagnosis of DNA samples extracted from cells This technique is an effective method to detect healthy people carrying disease genes in Vietnam The success of this technique opens up a new application in reducing the birth of sick children by pre-implantation diagnosis counseling The study was conducted with 35 hemophilia A families with 77 female members, without using point mutations but only on clinical diagnosis Use of microsatellite DNA techniques identifies mutant alleles The results showed that 35/35 mothers with mutant genes in heterozygous state accounted for 100% and 68/77 female members were healthy people carrying mutated genes F8 35/35 mothers carry disease genes (accounting for 100%) The results of determining the maternal status of the mother are very important: if the mother with the disease gene can transmit the disease to the son and transmit the disease to their daughter, the disease can be inherited Over generations and many people have been sick in the same family In no case, a mother who does not carry the disease, the mutation of the F8 gene in a sick child cannot be a mosaic or a new denovo mutation, the female members of the patient's family are all risk of carrying disease genes 68/77 healthy people carry disease genes on 35 hemophilia A patients (88.3%), which is higher than the study of Shetty S (2001) on 102 families of hemophilia A (India) patients The rate of healthy people carrying disease genes is 64.5%, higher than that of Bui Thi Thu Huong (2014) on 50 families of hemophilia A patients seeing a proportion of healthy people carrying disease genes is 53.6% This difference can be attributed to the fact that the results obtained on the sample size are small so it cannot be concluded, it is also possible that the subjects in the studies had different medical histories, which the female family members had The more obvious a medical history is, the higher the risk of carrying the disease In this study our female members of the hemophillia A family who have not yet fully participated in the study may have female members of the family who are still inferior or fearful, so they have not yet checked to determine status of carrying genes With 68 people 24 carrying disease genes should be counseled and genetic counseling, preventing the birth of children with hemophilia A disease, increasing effectiveness in preventing disease The determination of healthy people carrying disease genes is very important, because if the mother carries the disease, the mother has hereditary characteristics, transmitting the disease to the son, manifesting the disease in clinical The research shows that women with disease genes need to have a contingency plan to ensure general health and reproductive health in particular to improve the quality of life themselves, families and society As a result, there are women who not carry the disease, which will help women to be completely free of disease obsession and anxiety in life The woman with motherhood is very hard, the women who carry the hemophilia A gene also have a lot of obsession, anxiety and hardship, taking care of the birth of a sick and difficult child in childbirth due to the risk of obstetrical bleeding However, there are difficulties that women who not know they carry the disease due to lack of knowledge about the disease and are not propagandized for marriage counseling Although this is not the first molecular biology technique to identify healthy people carrying disease genes In 2014, Bui Thi Thu Huong and colleagues used I-PCR technique to detect healthy people carrying disease genes based on point mutations, this technique gives accurate results However, this technique must rely on sequencing on patient-based mutations as a difficult, hard and expensive technique While families of hemophilia A patients are difficult families, the implementation of the I-PCR technique to detect mutations is limited So microsatellite DNA technology was developed to help hemophilia A patients with higher performance because this technique is quick and simple to analyze hemophillia A in families that cannot be identified mutations The results of pre-implantation diagnosis The discovery of healthy people carrying disease genes is an important basis for conducting prenatal diagnosis to detect early fetuses carrying disease genes, reducing the birth of sick children With prenatal screening performed by amniocentesis at 15-18 weeks of pregnancy, it 25 was studied by I-PCR technique However, with pregnancy screening, the disadvantage is that if the fetus is identified, it will have to stop the pregnancy when the pregnancy is too large, affecting the physiological mind of the mother, affecting the quality of the fetus the following pregnancies Therefore, pre-implantation diagnosis technique helps couples to be able to produce completely healthy children without having to suspend pregnancy Subjects needing pre-implantation diagnosis in our study were women who had a disease gene that had or had not had a child with hemophillia A had been identified to carry disease genes in heterozygous state by microsattellite technique DNA The study carried out on 75 embryonic stem cells, resulting in 49/75 (65.3%) normal embryos, 26/75 (34.7%) abnormal embryos This shows that if there is no genetic diagnosis before nesting, the probability of having sick children in the community is 34.7%, which is a burden for families and society Thus, along with the development of the country, molecular biology techniques are increasingly developing, from the discovery of direct gene mutations in patients, to early diagnosis of pregnant women in the second week of pregnancy 15 affecting the quality, physiological psychology of women, today microsatellite DNA technology was born that solved the limitations of those methods So it is not difficult to give birth to a strong child on families with people with hemophilia A For families with difficult children, the desire to have a very large child, although the biopsy is the embryo that carries the disease gene These embryos, after growing up, will definitely have to consult with genetic diagnosis of prenatal diagnosis and pre-implantation diagnosis CONCLUSION From the research results, some conclusions can be made: Detecting healthy people carrying F8 gene mutations by microsatellite DNA technique In 35 families with children with hemophilia A have been clinically diagnosed: - 35/35 mothers with mutated F8 gene in heterozygous state, accounting for 100% 26 - 33/42 female members (including grandmother, uncle, aunt, sister, sister ) carry disease genes in heterozygous state, accounting for 78.6%; 9/42 female members not carry disease genes, accounting for 21.4% Pre-diagnosis of nesting of hemophilia A by microsatellite DNA technique 12/35 pregnant women who are healthy with disease genes are preimplantation diagnosis - The total number of embryos tested is 75 embryos, of which the abnormal embryonic genes account for 34.7%, the number of normal embryos accounts for 65.3% - 9/12 patients mothers have normal embryos after genetic analysis The research results are solid scientific evidence that microsatellite DNA technology is a highly reliable and suitable technique for families of hemophilia A patients who wish to identify healthy people with disease genes, families who wish to give birth by pre-implantation diagnosis ... F8 gene mutations cause disease In the endogenous coagulation pathway, factor VIII acts as a cofactor with activated factor IX (IXa) in the presence of Ca2 + and platelet phospholipid activating... time), normal fibrinogen, APTT (Activated Partial Thromboplastin Time: partially activated thromboplastin time) lasts> 1.5 normal values + The activity of plasma VIII factor decreases below 40%... conditions: Have a father with hemophilia A disease; At least two 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 to hemophilia