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MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY - VU THI BICH HUONG STUDY ON MUTATIONS AND GENETIC POLYMORPHISMS OF THE IX FACTOR CODING GENE IN HEMOPHILIA B PATIENTS Specialty: Genetics Code: 42 01 21 SUMMARY OF PH.D THESIS Ha Noi - 2022 Study was completed in Graduate University of Science and Technology – Vietnam Academy of Science and Technology Advisor 1: Dr Duong Quoc Chinh National Institute of Hematology and Blood Transfusion Advisor 2: Assoc.Prof.Dr Dong Van Quyen Institute of Biotechnology, Vietnam Academy of Science and Technology Reviewer 1: Assoc.Prof.Dr Vu Minh Phuong Reviewer 2: Assoc.Prof.Dr.Luong Thi Lan Anh Reviewer 3: Assoc.Prof.Dr Vu Thi Thom The thesis will be defended in front of the University level doctor thesis assessment Committee, taking place in Graduate University of Science and Technology – Vietnam Academy of Science and Technology at … … , date … month … year … The thesis is available at: - Library of Graduate University of Science and Technology - National Library of Vietnam FOREWORD Hemophilia B is a bleeding disorder caused by deficiency of factor IX (FIX) This is a persistent condition, which usually causes severe consequences and threatens patients’ lives The pathogenic mechanism of hemophilia B is due to mutation of the factor IX coding gene (F9), which leads to decrease or loss of FIX synthesizing function of genes, leading to deficiency of factor IX, resulting in prolonged bleeding F9 is a recessive gene on the X chromosome which is inheritable through generations Therefore, carrier diagnosis is the only way to control the source of defective genes, limit the rise of new cases via prenatal diagnosis and preimplantation genetic diagnosis Mutations and nucleotide polymorphisms on F9 gene are the main markers for hemophilia B diagnosis These genetic changes are population related, thus, it is important to identify the causative mutation and informative polymorphisms in each population, to establish effective and suitable diagnostic protocol Research on mutations and genetic polymorphisms of the hemophilia gene F9 has been conducted for a long time worldwide Many countries have published F9 gene mutation spectrum and nucleotide polymorphisms in their own populations, contributing to treatment, control and management hemophilia B In Vietnam, there were several studies conducted regarding F9 gene mutation However, those studies were limited in either small number of patient or genetic markers in F9 gene Therefore, it is lacking a systematic study on mutations and nucleotide polymorphisms on the F9 gene for Vietnamese population As the number of new cases increases every year, a crucial mission to control defective gene sources is to conduct a population study on F9 gene with a large sample size With all those reasons, we conducts the study, entitled: “Study on mutations and genetic polymorphisms of the factor IX coding gene in hemophilia B patients” Objective of the study: (1) To identify the mutation on the factor IX coding gene of Vietnamese hemophilia B patients (2) To determine nucleotide polymorphisms that have a high rate of heterozygosity as specific genetic markers for gene carrier diagnosis by linkage analysis method Main contents of the study: (1) Building F9 gene sequencing protocol on NGS system (2) Sequencing and mapping F9 mutation in 100 patients Examining the relation between mutation genotype and disease severity (3) Sequencing the F9 gene in 100 normal females Determining the frequency and rate of heterozygotes of nucleotide polymorphisms on the F9 gene CHAPTER LITERATURE REVIEW 1.1 Synopsis of hemophilia B 1.1.1 Some historical mark about discovery and research on hemophilia B The first report about clotting difficulty appeared in the second century AD In 1928, the term “hemophilia” was first mentioned In 1952, Rosemary Biggs discovered the disease hemophilia B In 1982-1983, the F9 gene was cloned In 1985, the sequence of the F9 gene was published From here on, genetic analysis for hemophilia B gene carrying conditions had been conducted 1.1.2 Overview of hemophilia B 1.1.2.1 Characteristics A normal healthy person has a concentration of active FIX ranging from 50 - 150% A hemophilia B patient loses the ability to control the coagulation process due to active factor IX concentration reduced to under 40% 1.1.2.2 Genetic mechanism Hemophilia B is recessively linked to the X chromosome About 70% of cases are due to mutated X chromosomes inherited through generations in a family, 30% is due to a de novo mutation 1.1.2.3 Diagnosis and treatment method a) Detection diagnosis Quantitative test of factor IX combining with clinical diagnosis and examining family history b) Severity diagnosis Severe (FIX < 1%), moderate (FIX - %), mild (FIX - 40%) c) Treatment Mainly infusion of exogenous factor IX New treatment methods including gene therapy are in clinical trials 1.2 Structure and function of FIX in coagulation process 1.2.1 Coagulation factors Substances joining the coagulation process include serous protein, tissue factors (TF), ion Ca2+, membrane phospholipid and platelet cells Substances that promote coagulation processes are coagulation factors, including factor IX 1.2.2 Role of factor IX in coagulation process factor IX has a pivotal role in activation of X factor to become factor Xa, which is the final target of the coagulation pathway 1.2.3 Structure and activation process of factor IX The F9 gene is approximately 34 kb, including exons and introns Factor IX molecules are synthesized in the liver, weighing 57kDa, with domains: GLA, EGF1, EGF2, active peptide and catalyst domain 1.2.4 Interaction between FIX and activation factor Domain SP is the link location between factor IXa and factor VIIIa Domain EGF (EGF1, EGF2) has a crucial role in the interaction between factor IXa and factor VIIIa Domain GLA supports the molecule to link with phospholipid surface of activated platelets 1.3 Genetic analysis of hemophilia B 1.3.1 The role of genetic analysis in hemophilia B Genetic analysis for detection of carriers and prenatal diagnosis, preimplantation genetic diagnosis for females in families with hemophilia B is effective, and the only way to control defective gene sources 1.3.2 Traits of F9 gene mutations 1.3.2.1 Single nucleotide polymorphisms Single nucleotide substitution is the most common one among hemophilia B mutations, accounting for 64%, including missense, nonsense or mutations, or mutations at intron-exon boundaries 1.3.2.2 Founder effect mutations The phenomenon in which some mutations occur at high ratios in some populations, unique to them It may be due to CpG switch or inbreeding 1.3.2.3 Mutation leading to hemophilia B Leyden Mutation occurs in the regulatory region Patients show decrease in FIX concentration before puberty, but increase to close to normal since puberty 1.3.2.4 Small deletions, insertions DNA repricationmistakes leading to point mutations, and nucleotide insertion or deletion These mutations usually occurred in nucleotide-repeated sequences, especially at locations in which repeated sequences are adjacent 1.3.2.5 Large genetic abnormality Accounting for 3-5% of mutations in hemophilia B This includes rearrangement of gene, loss of large fragment of gene (loss of or many exons, or even loss of whole F9 gene) 1.3.3 Traits of nucleotide polymorphisms linked to F9 gene 1.3.3.1 Overview of nucleotide polymorphisms Changes of nucleotides (SNP or VNTR) which may or may not change coding triplet but not change structures, functions of genes and they are not mutations, but are valuable in diagnosis Informative polymorphisms and uninformative polymorphisms Informative polymorphism is a polymorphism with different alleles (heterozygote) of the locus Polymorphism with identical alleles (homozygote) of the locus is an uninformative polymorphism, having no value in linkage analysis Intragenic marker and extragenic marker Polymorphisms inside the F9 gene are called intragenic markers and polymorphisms outside of the F9 gene are called extragenic markers Only intragenic markers should be used in linkage analysis for carrier screening Linkage equilibrium polymorphisms and linkage disequilibrium polymorphisms Two polymorphisms which always inherited together are called complete linkage disequilibrium, while those independently inherited are called linkage equilibrium Using linkage equilibrium polymorphisms will increase diagnosis efficiency 1.3.3.2 Traits of polymorphisms on F9 gene F9 gene mostly has single nucleotide polymorphisms, there only have been repeated polymorphisms (RY)n at intron and exon that were discovered The ratios of alleles are different between populations, these differences need to be researched in detail in order to apply linkage analysis effectively 1.3.3.3 Ratio and heterozygosity of polymorphisms The rate of polymorphic alleles occurrence is rarely balanced, but usually one allele will have dominance over the others Data about allele frequency allows calculation of indices of genetic polymorphisms such as heterozygosity, in theory 1.4 Genetic analysis techniques in hemophilia B 1.4.1 Mutation analysis techniques 1.4.1.1 Southern blot hybridization The first technique used in mutation analysis of hemophilia B However, due to it’s complexity, time consuming, and requiring toxic chemicals, this technique is not widely used nowadays 1.4.1.2 Heteroduplex analysis Mutation screening based on heteroduplex is conducted to localized gene regions suspected with mutation Then, these regions are sequenced to determine genetic changes 1.4.1.3 Sanger sequencing Focusing on analysis of exons, intron-exon junction, 5’UTR and 3’UTR of F9 gene This procedure has been standardized and applied in many laboratories worldwide 1.4.1.4 Next Generation Genome Sequencing (NGS) NGS is the new method in hemophilia B mutation analysis NGS can analyze the whole F9 gene including all exons and introns, is very effective at mutation analysis and finding informative polymorphisms in population study 1.4.1.5 Large deletion detection technique MLPA technique is used to detect unknown large gene deletions, loss of a part of an exon, loss of some exons or loss of the whole F9 gene 1.4.2 Linkage analysis technique Based on nucleotide polymorphisms in the F9 gene to trace the mutated alleles in patients’ families This method can not directly clarify the mutation but can indirectly diagnose carrier status of family members 1.5 F9 gene polymorphisms research status 1.5.1 Research status worldwide The two main approaches to genetic analysis in hemophilia B is to directly analyze mutation and to indirectly analyze via F9 linked polymorphisms Research about hemophilia B causing mutation and traits of F9 linked polymorphisms are deployed strongly Many countries have published the traits of mutations and nucleotide polymorphisms of their own countries’ populations 1.5.2 Research status in Vietnam Statistics of Hemophilia Center, National Institute of Hematology and Blood Transfusion shows that there are hundreds of new cases every year, and the number of carriers cannot be controlled yet Diagnosis, management and treatment still face many challenges, shortage of therapeutic substances, decrease of patients’ life standard, high impairment rate Therefore, prevention work becomes very crucial However, it is still lacking about a systematic study on nucleotide polymorphism and mutation characteristics of F9 gene, except some small research Thus, the published data is insufficient for genetic counseling, prenatal and preimplantation diagnoses and prevention of newborn affected cases CHAPTER SUBJECTS AND METHODS OF THE STUDY 2.1 Subjects of the study 2.1.1 Patient samples: 100 hemophilia B patients diagnosed and treated in National Institute of Hematology and Blood Transfusion (sample labeled: HB01HB100) (for goal number 1: F9 gene mutation mapping) 2.1.2 Healthy people sample: 100 healthy females participating in blood donation in National Institute of Hematology and Blood Transfusion (sample labeled: NK01-NK100) (for goal number 2: screening nucleotide polymorphisms on F9 gene) 2.1.3 Carrier sample: 23 females who carry the defective gene (MG1-MG23) (for assessing effectiveness of polymorphism set - the result of goal number 2) - Each subject was drawn ml of peripheral blood (anti clot by EDTA) 2.2 Methods: 2.2.1 Study design Cross-sectional descriptive study with analysis 2.2.2 Formula for sample size calculation in nucleotide polymorphism study Leslie Kish formula: n = (Z1-α)2 [p (1-p)/D2]) 2.2.3 Study diagram 2.3 Chemicals and apparatus 2.4 Techniques used in study 2.4.1 Sample collection Each study sample was drawn ml of blood into EDTA tube Blood drawing procedure was ensured completely disinfected 2.4.2 DNA extraction of study samples Using Qiagen’s DNA extraction kit 2.4.3 Designing primer sequences for F9 gene amplification Using Primer3 software to design the primer pairs Testing the F9 gene coverage of primer sequences on CLC Genomic Workbench software 2.4.4 Amplification of F9 gene by Longrange PCR (LR-PCR) Optimized LR-PCR procedure to amplify F9 gene regions using Q5 HighFidelity 2x Mastermix kit 2.4.5 Electrophoresis of PCR products on agarose gel 2.4.6 Purification of amplified F9 gene regions PCR products Using Agencourt Ampure XP kit 2.4.7 Sequencing F9 gene by NGS technique 2.4.7.1 Preparing DNA library: using Nextera XT Library Prep kit 2.4.7.2 Purifying PCR product library: Using Agencourt Ampure XP kit 2.4.7.3 Standardizing sample: measure the concentration of libraries using QuBit dsDNA HS Assay kit, converting units from ng/µl to nM 2.4.7.4 F9 gene sequencing: Using Miseq Reagent kit 2.4.8 Confirming F9 gene sequencing results on NGS by Sanger sequencing technique 2.4.8.1 PCR-Bigdye reaction: Using BigDye™ Terminator v3.1 kit, following manufacturer’s instruction 2.4.8.2 Purifying PCR-Bigdye products: Using Bigdye Xterminator kit following manufacturer’s instruction 2.4.8.3 Sequencing reaction on AB 3500 2.4.9 Confirming deletion/duplication on F9 gene Using MLPA to confirm cases with suspicion of large deletion, using SALSA MLPA P207 F9, MRC Holland kit following manufacturer’s instruction 2.4.10 Analyzing mutation from sequencing data Using Miseq Reporter and CLC Genomics Workbench 9.5.2 softwares; referencing genes are NG_007994.1 and NM_000133.3; referencing database belongs to EAHAD (http://www.factorix.org), CDC (https://www.cdc.gov/ncbddd/hemophilia/champs.html) and international SNP database (https://www.ncbi.nlm.nih.gov/snp) - Using PolyPhen-2 and SIFT softwares to predict impact of new changes to structures and function of F9 gene 2.4.11 Analyzing nucleotide polymorphisms on F9 gene Using CLC Genomic Workbench software; reference sequence F9_NC000023.11; NCBI SNP database (https://www.ncbi.nlm.nih.gov/snp) Calculating polymorphism indices: consisting of polymorphed alleles frequency, heterozygote rate of polymorphs, link between SNP (on Haploview 4.2 software) Confirming polymorphism set effectiveness: by applying the above analysis on 23 gene carrying females, then calculating number of heterozygote cases when combining polymorphism set CHAPTER RESULTS AND DISCUSSION 3.1 Sample preparation 3.1.1 Sample collection Samples were collected from 100 healthy unrelated people, with average age at 24 ± 14, and no excessive bleeding history; 100 samples of hemophilia B patients as shown in Table 3.1 Table 3.1 Traits of study’s patients Hemophilia B patients (n=100) Characteristics n Male 99 Sex Female Severe 79 Hemophilia B severity Moderate 17 Mild Yes Status of Inhibitors No 100 Average age (x ± SD) 22 ±16 % 99 79 17 100 Patients in study were mostly males (99%) 79% were severe (FIX concentration < 1%), 17% were moderate (FIX concentration -5 %), 4% were mild (FIX concentration - 40%) All patients in study did not have FIX inhibition in the treatment period 3.1.2 Materials preparation High concentrated DNA samples (>100 ng/µl) were obtained indicating as intact DNA bands (Fig 3.1 and Table 3.2) Figure 3.1 Results of total DNA extraction HB1-HB6: patient (1 - 6) NK1-NK6:6 healthy females (1-6)M: DNA ladder 1kb (Thermo Fisher) Table 3.2 Concentration result and purity of some DNA samples No Sample Concentration A260 No Sample Concentration A260 (ng/µl) /A280 (ng/µl) /A280 NK1 90 1.95 108.8 1.9 HB1 HB2 94 1.91 NK2 93.6 1.94 HB3 150.1 1.91 NK3 111.7 1.9 HB4 108.1 1.9 10 NK4 119 1.88 HB5 93 1.91 11 NK5 207.5 1.9 HB6 225.6 1.88 12 NK6 207.5 1.91 11 Stability of optimized ingredients and heat cycle were confirmed with repeated reactions on 10 patients Thus, by LR-PCR at above heat cycles, we amplified the whole 35kb long-F9 gene (Fig 3.9) Figure 3.9 Amplification images of a1-a8 at optimized Tm M: Thang ADN chuẩn kb (Thermo Fisher) a1- a8: phân đoạn a1 -a8 gen F9 LR-PCR procedure with optimized ingredients and conditions was used to amplify a1-a8 regions for all study samples The products of LR-PCR were then purified and used to prepare a DNA library to sequence the F9 gene by NGS 3.2.3 DNA library preparation results Using Nextera XT DNA Library preparation kit, we yielded the library including DNA with size mostly at about 462 bp, in the standard zone for DNA library which is from 300 - 500 bp (Fig 3.10) Figure 3.10 Result of DNA library electrophoresis on BiOptic system 3.2.4 Results of sequencing with optimized NGS procedure The DNA library was purified by Agencourt Ampure XP, then sequencing was conducted with Miseq Reagent kit Results showed that quality indices met technical criteria, with Q30 = 90,5%; density = 1153 K/mm2; Cluster passing filter = 88,2% (Fig 3.11a) Collected sequencing data covered the whole F9 gene (35kb) (Fig 3.11b) Minimum coverage for each base was 100X 12 Figure 3.11a: Quality of NGS Q30 = 93,8%; density = 896 K/mm2; Cluster passing filter = 91,7% Figure 3.11b: Some results of NGS Figure 3.11b: Some results of NGS 3.2.5 Confirming the NGS results Reliability of NGS procedure was confirmed when the results of parallel sequencing of 10 patients’ samples with NGS and Sanger showed 100% similar mutation analysis results (Fig 3.12) Result of NGS Result of Sanger sequencing (1) HB04 c.128G>A c.128G>A (2) HB05 c.382T>C c.382T>C (3) HB06 c.676C>T c.676C>T 13 (4) HB08 c.689G>T c.689G>T (5) HB12 c.127C>T c.127C>T (6) HB15 c.677G>A c.677G>A (7) HB18 c.470G>T c.470G>T (8) HB19 c.1135C>T c.1135C>T (9) HB22 c.874C>T and c.933G>C c.874C>T and c.933G>C (10) HB25 c.956T>C c.956T>C Figure 3.12 Results of parallel sequencing of 10 patients’ samples with NGS (right) and Sanger (left) 14 3.3 F9 gene sequencing results in studied samples Using the NGS procedure established above, we successfully sequenced the F9 gene for 200 study samples (100 patients and 100 healthy females) Except for data of patients labeled H14, H94, H97, data from the other 197 samples (97 patients and 100 healthy females) all covered the whole F9 gene (35kb), meeting the criteria for analyzing genetic changes (including mutations and nucleotide polymorphisms) on the F9 gene Sample H14, H94, H97 lost Longrange PCR products at region a5 (H14), a2-a8 (H94), a4-a6 (H97) due to gene deletion (Fig 3.13), therefore only the data of remaining gene regions were collected (Fig 3.14) All these samples were confirmed to have deletion by MLPA technique (Fig 3.15) H14: Failure of amplification of a5 H94: Failure of amplification of a2-a8 H97: Failure of amplification of a4-a6 Figure 3.13 Results of Longrange PCR of H14, H94, H97 H14: Loss of exon H97: Loss of exon -6 H94:loss of exon - Figure 3.14 Result of NGS of H14, H94, H97 H14: del- exon H94: del_ exon - H97: Del_ exon -6 Figure 3.15 Results of MLPA of H14, H94, H97 3.4 Results of mutation analysis of 100 study patients NGS data showed that 100% patients have disease-causing mutations (Appendix 01) The study discovered 58 different mutations, including 16 new mutations New mutation ratio was 27,59% List of 58 mutations is shown in table 3.8 15 Table 3.8 List of 16 new mutations in the current study 3.5 Traits of mutation found in this study 3.5.1 Types of mutation types of mutation were found (point mutation, insertion mutation, small deletion, large deletion) Point mutation was the most prevalent (91,38%) Table 3.10 Traits of mutation found in this study Type of mutation Missense Insertion Small deletion (50 bp) Total n 53 1 58 % 91,38 1,72 1,72 5,17 100 16 Table 3.9 List of 58 mutations in the current study *: The mutation are confirmed by MLPA 17 3.5.2 Impact of mutations on structure and function of F9 gene Mutations found were diverse, affecting one to many nucleotides, some mutations caused the loss of 1-2 exons, or even exons of the F9 gene Point mutations leading to new amino acids accounted for the highest ratio, at 75,47% Some mutations led to frame shift, making nonsense codons or splicing variations (Table 3.11) Table 3.11 Impact of mutations on structures and functions of gene Type of mutation Point (n = 53) Insertion (n = 1) Large deletion (n=3) Small deletion (n = 1) Effects of mutation n % Missense 40 75,47 Nonsense 9,43 Splice site 9,43 Frameshift 3,77 Promoter Frameshift 1,89 - Loss exon - Loss exon - Loss exon Loss codon - - 3.5.3 Recurrent mutations in study patients We found 16/58 mutations occurred in at least patients The most was c.881G>A, occurred 12 times (Table 3.12) Table 3.12 List of recurrent mutations in study patients 18 3.6 Associations between severity and mutation type In this study, large genetic damages such as deletion, nonsense mutation, frameshift mutation only occurred in severe patients Missense mutation occurred at all groups of severity (Table 3.13) Table 3.13 Relation between mutation type and disease severity Type of mutation Missense Frameshift Nonsense Small deletion Large deletion Splice site Promoter Total (n = 100) Severe (FIX 5 – 40%) N % 100 0 0 0 0 0 0 3.7 F9 gene mutation characteristics of hemophilia B patients in Vietnam 3.7.1 Distribution of mutation on F9 gene Mutations found in this study spread from the beginning to the end of F9 gene, with the most was at exon (46,55%), followed by exon (12,07%); exon did not have mutation mutations were found in intron 2, 3,5,7 Rate of mutation occurred in exon was 89,66% (52/58 mutations); in intron and 5’UTR region was 10,34% (6/58 mutations) (Figure 3.16) Figure 3.16 Distribution of mutations on F9 gene Figure 3.17 Distribution of mutations on FIX domains 3.7.2 Distribution of mutations on FIX domains Mutations mostly distributed on domain SP (51,72%), the rate of mutation on other domains ranged from 3,45 % - 8,62% In one case (1,72%) the mutation caused the loss of domain EGF1-EGF2-AP, in one case the mutation caused the loss of all domains of FIX molecule (Figure 3.17) 19 3.7.3 Map of F9 gene mutation of hemophilia B patients in Vietnam From the mutation data collected, we made the map of the F9 gene in hemophilia B study patients The map shows all 58 mutations found in the study Location of occurrence, rate of mutation in each exon, intron of F9 gene or in domains of FIX molecule are shown clearly, generalizing the traits genetic damages on F9 gene in hemophilia B patients in Vietnam Figure 3.18 Map of F9 gene mutation of hemophilia B patients 3.8 Analysis of nucleotide polymorphisms on F9 gene 3.8.1 Nucleotide polymorphisms screening on factor IX coding gene Analyzing NGS data from 100 healthy females, we discovered 12 potential SNP with heterozygote rate ≥ 29% (Table 3.14) Table 3.14 List of 12 SNPs discovered from sequencing data SNP HGVS name (NM_000133) Position on reference gene NC_000023.1 GRCh38.p1 NC_000023.11:g.13952810A >C 139528190 c.-822A>G c.-727T>C c.88+75A>G c.88+1660T>C c.88+251oC>T c.89-2527C>A c.89-2518T>C c.89-2368A>T c.89-1859C>G c.278-981T>C c.520+565G>A Reference Allele Change Allele Outside A C 139529943 139530038 5’UTR 5’UTR 139530927 139532512 139533362 intron intron intron intron intron intron intron intron intron A T A T C C T A C T G G C G C T A C T G C A 139534483 139534492 139534642 139535151 139540095 139549056 Position on F9 SNP code rs20167974 rs411017 rs378815 rs3817939 rs401597 rs371000 rs4149670 rs4149671 rs4149674 rs392959 rs374988 *Light gray color highlights the new SNP in the current study F9 gene is located from139530720 to139563459 on X chromosome, NC_000023.1 Heterozygosit y frequency (%) 41 41 41 43 41 29 29 29 29 48 47 30 20 All 12 polymorphisms were SNP; there were SNPs in introns, SNP in 5’UTR of F9 and SNP outside of F9 gene Among those, 11 SNP had been published, SNP were newly found in this study (c.89-1859C>G) - this is the SNP with the highest heterozygote rate (48%) 3.8.2 Rate of polymorphic alleles in healthy population Screening results showed that the referenced allele is the common allele at all 12 SNPs The rate of minor allele ranged from minimum at 16 % (polymorphed locus c.89-2527C>A, c.89-2518T>C, c.89-1859C>G) to maximum at 36% (polymorphed locus c.278-981T>C, c.520+565G>A) (Table 3.15) Table 3.15 Frequency of polymorphed alleles in the study Allele frequency (n = 300) ID number Nucleotide polymorphism NC_000023.11:g.139528190A>C c.-822A>G c.-727T>C c.88+75A>G c.88+1660T>C c.88+2510C>T c.89-2527C>A c.89-2518T>C c.89-2368A>T 10 c.89-1859C>G 11 c.278-981T>C 12 c.520+565G>A Allele n % A C A G T C A G T C C T C A T C A T C G T C G A 222 78 202 98 202 98 230 70 199 101 253 47 253 47 253 47 253 47 252 48 192 108 193 107 74 26 67 33 67 33 77 23 66 34 84 16 84 16 84 16 84 16 84 16 64 36 64 36 3.8.3 Analyzing the link between nucleotide polymorphisms Our study found out that the SNP group: rs201679740, rs411017, rs378815, rs401597 rs371000, rs4149670, rs4149671, rs4149674 almost always occurred together in all research samples; 27/30 (90%) of s374988 polymorphisms always with rs392959 (Table 3.16) 21 Table 3.16 : List of co-occurred nucleotide polymorphisms 22 Analysis results of the link between SNPs on Haploview software also showed that rs371000, rs4149670, rs4149671, rs4149674 polymorphisms were fully linked with D’ = Rs374988 and rs392959 polymorphisms were strongly linked with D’ = 0,79 The link between the two polymorphisms Rs3817939 and c.89-1859C>G with other SNPs were ranging from low to moderate (D’ = 0,14 0,54) (Figure 3.17) D’ (%) Figure 3.17 Analyzing the link between nucleotide polymorphisms on Haploview 4.2 3.8.4 Choosing the informative polymorphism set Based on the analysis results of the link between SNPs, we divided 12 SNPs into groups based on their degree of linkage (Table 3.18) Bảng 3.18 Grouping SNPs based on degree of linkage 23 From that, we established a set of polymorphisms consisting of SNPs, including SNPs standing for SNP groups with strong to full link (group 1, 2, 3) and SNPs group (Table 3.17) Table 3.17 Informative SNP set STT SNP HGVS name (NM_000133) Position on reference gene NC_000023.11 GRCh38.p1 Position on F9 Reference Allele Change Allele SNP code Heterozygosity frequency (%) c.-727T>C 139530038 5'UTR T C rs378815 41 c.89-2527C>A 139534483 intron C A rs4149670 29 c.278-981T>C 139540095 intron T C rs392959 47 c.88+75A>G 139530927 intron A G rs3817939 43 c.89-1859C>G #VALUE! intron C G 48 3.8.5 Confirming effectiveness of polymorphism set in patients’ families Effectiveness of set SNP was confirmed when analyzing 23 samples, of whom carrying the defective gene, it was shown that 23/23 (100%) had at least one heterozygous SNP The rate of diagnosis in combination was 100% There were heterozygous samples with SNP (17,39%), heterozygous samples with SNPs and SNPs (26,09%), heterozygous samples with SNP (21,74%), heterozygous samples with all SNPs Individual diagnosis effectiveness with each of the following SNP: rs378815, rs3817939, rs4149670, c.89-1859 C>G, rs392959 were respectively 60,87% (14/23); 56,52% (13/23); 39,13% (9/23), 69,57% (16/23) 30,43% (7/23) (Figure 3.20) Figure 3.20 Confirming effectiveness of polymorphism set 24 CONCLUSION AND RECOMMENDATION CONCLUSION Successfully mapped 58 different mutations from 100 Vietnamese hemophilia B patients Total number of different mutations found in this study was 58, including 16 new mutations.The mutation map showed in detail all traits, as well as location of occurrence, rate of mutation emerging in exons and introns, rate of mutation in each exon and intron specifically, distribution of mutations in domains of F9 gene of all 58 discovered mutations Have screened the nucleotide polymorphisms on the whole F9 gene from 200 studied samples The marker set consisting of polymorphisms has been built: c.89-1859 C>G, rs392959, rs3817939, rs378815, rs4149670 with high heterozygote rate, from 29% - 48% Applicability of the polymorphism set reached 100% in analysis on 23 gene carrying people, corresponding to 23 families of hemophilia B patients RECOMMENDATION Use the mutation database collected from this study for diagnosis of defective gene carriage via mutation analysis Applying the polymorphism set c.89-1859C>G, rs392959, rs3817939, rs378815, rs4149670 on larger number of gene carrying people to give reasonable advise about the order of using SNPs NEW CONTRIBUTION OF THE STUDY This is the first and systematic study in Vietnam about mutations on the whole factor IX coding gene causing hemophilia B with a large sample size, including all functional regions, exons and introns, using NGS technique This is the first study in Vietnam about nucleotide polymorphisms on factor IX coding gene, on large sample size, serving as gene carrier diagnosis by link analysis LIST OF PUBLISHED RESEARCHES Khanh Q Bach, Chinh Q Duong, Huong B.T.Vu, Anh.T.Tran (2022) Mutation characteristics of hemophilia A and B patients in the National Institute of Hematology and Blood Transfusion, Ha Noi, Viet Nam World Federation of Hemophilia 2022 (WFH 2022, World Congress ) Vu Thi Bich Huong, Tran Tuan Anh, Nguyen Thanh Ngoc Binh, Nguyen Thi Mai, Bach Quoc Khanh, Duong Quoc Chinh (2022) Application of nextgeneration sequencing to investigate F9 polymorphism Vietnam Medical Journal 511: 242 – 247 Duong Quoc Chinh, Vu Thi Bich Huong, Tran Tuan Anh, Nguyen Le Anh, Ngo Thu Hang, Nguyen Thi Mai, Nguyen Ha Thanh, Bach Quoc Khanh (2020) Develop techniques to complete molecular diagnostic regimen for hemophilia at National Institute of Hematology and Blood Transfusion Vietnam Medical Journal 496: 802 – 815 Vu Thi Bich Huong, Nguyen Le Anh, Tran Tuan Anh, Bui Thuy Huong, Ninh Thi Thanh Thao, Vu Quang Lam, Nguyen Thi Mai, Bach Quoc Khanh, Dong Văn Quyen, Duong Quoc Chinh (2018) Combination of direct analysis and linkage analysis to detect hemophilia gene carrier at the national institute of hematology and blood transfurion Vietnam Medical Journal 466: 1085 – 1090 Duong Quoc Chinh, Vu Thi Bich Huong, Nguyen Le Anh, Tran Tuan Anh (2018) Application of Next-Generation Sequencing to analyze F9 gene mutation in hemophilia B patients Vietnam Medical Journal 466: 581 – 590 Vu Thi Bich Huong, Nguyen Le Anh, Tran Tuan Anh, Bui Thuy Huong, Ninh Thi Thanh Thao, Vu Quang Lam, Nguyen Thi Mai, Bach Quoc Khanh, Dong Văn Quyen, Duong Quoc Chinh (2018) Initial study of markers MseI, HhaI, TaqI, XmnI, DdeI in a group blood donors at the nationation insitute of hematology and blood transfusion Vietnam Medical Journal 466: 591 – 600 ... mutations and genetic polymorphisms of the factor IX coding gene in hemophilia B patients” Objective of the study: (1) To identify the mutation on the factor IX coding gene of Vietnamese hemophilia. .. Biggs discovered the disease hemophilia B In 1982-1983, the F9 gene was cloned In 1985, the sequence of the F9 gene was published From here on, genetic analysis for hemophilia B gene carrying conditions... mechanism of hemophilia B is due to mutation of the factor IX coding gene (F9), which leads to decrease or loss of FIX synthesizing function of genes, leading to deficiency of factor IX, resulting
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