Study on the association between tdrd1 rs541192490 and male infertility in a vietnamese population

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY 🙡 🕮 🙣 UNDERGRADUATE THESIS “STUDY ON THE ASSOCIATION BETWEEN TDRD1 rs541192490 AND MALE INFERTILITY IN A VIETNAMESE POPULATION” HANOI – 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY 🙡 🕮 🙣 UNDERGRADUATE THESIS “STUDY ON THE ASSOCIATION BETWEEN TDRD1 rs541192490 AND MALE INFERTILITY IN A VIETNAMESE POPULATION” STUDENT : NGUYEN MINH NGUYET CLASS : K62CNSHE MAJOR : BIOTECHNOLOGY SUPERVISORS : NGUYEN THUY DUONG, PhD : TRAN THI HONG HANH, MSc HANOI – 2022 COMMITMENT I declare that this thesis has been composed solely by myself and has not been submitted, in whole or in part, for any other degree or professional qualification I confirmed that all methods, data, and results mentioned in this thesis are true and all references are fully cited according to standard reference practice Hanoi, February 22nd, 2022 Student Nguyen Minh Nguyet i ACKNOWLEDGEMENT I would like to express my deepest gratitude to my supervisor, Dr Nguyen Thuy Duong, who permitted me to work in Human Genomics Laboratory as a research student to conduct my undergraduate thesis While doing my thesis, she guided, supported and provided favorable conditions that helped me learn more knowledge in molecular biology, especially in research methods, then achieve targeted results I also want to thank MSc Tran Thi Hong Hanh for her monitoring and instruction to my study progression I want to appreciate my sincere to all staff at Human Genomic Laboratory for their assistance and mentoring Especially, I want to express my thanks to MSc Duong Thi Thu Ha for her careful and precious guidance, which was extremely valuable for my theoretical and practical study I would like to thank the Vietnam National University of Agriculture and Faculty of Biotechnology for providing a helpful chance to an undergraduate thesis at the Institute of Genome Research, which is a good opportunity to learn and experience after studying in the lecture hall Last but not least, I am strongly grateful for my family’s support and encouragement throughout my study journey Without them, my thesis can not be completed Student Nguyen Minh Nguyet ii CONTENTS COMMITMENT i ACKNOWLEDGEMENT ii CONTENTS iii LIST OF ABBREVIATIONS v LIST OF TABLES vi LIST OF FIGURES vii Part 1: INTRODUCTION Part 2: LITERATURE REVIEW 2.1 General concepts 2.2 Clinical evaluation 2.3 Causes 2.3.1 Physical and sexual factors 2.3.2 Environmental and lifestyle factors 2.3.3 Genetic factors 2.4 TDRD1 and male infertility 10 2.5 Principle of PCR-RFLP method for SNP detection 14 2.6 International and Vietnam research 16 2.6.1 International research 16 2.6.2 Vietnam research 17 Part 3: MATERIALS AND METHODS 19 3.1 Subjects 19 3.2 Materials 19 3.3 Methods 20 3.3.1 Total DNA extraction from whole blood samples 20 3.3.2 Primer design 21 3.3.3 Amplification of the target DNA region 21 3.3.4 PCR-RFLP 22 3.3.5 PCR Purification 23 iii 3.3.6 Sanger sequencing 24 3.3.7 Statistical analysis 25 Part 4: RESULTS 26 4.1 Total DNA extraction from whole blood samples 26 4.2 Amplification of the target DNA region 26 4.3 SNP Genotyping 27 4.4 Sanger sequencing 28 4.5 Association between TDRD1 rs541192490 and male infertility 29 Part 5: DISCUSSION 31 Part 6: CONCLUSION 33 REFERENCES 34 APPENDIX 47 iv LIST OF ABBREVIATIONS AZF Azoospermia factor CBAVD Congenital bilateral absence of the vas deferens CF Cystic fibrosis CFTR Cystic Fibrosis Transmembrane Conductance Regulator CI Confidence interval DNA Deoxyribonucleic acid FSH Follicle-stimulating hormone GWAS Genome-wide association study HWE Hardy-Weinberg Equilibrium LINE Long interspersed nuclear elements NOA Non-obstructive azoospermia OR Odds ratio PCR Polymerase chain reaction PiRNA Piwi-interacting RNA Piwi P-element-induced wimpy testis RE Restriction enzyme RFLP Restriction fragment length polymorphism SHBG Sex hormone-binding globulin SNPs Single nucleotide polymorphisms TDRD1 Tudor domain-containing WHO World Health Organization v LIST OF TABLES Table 2.1 The normal values for semen parameters over the years Table 3.1 The primer pair for amplification of TDRD rs541192490 19 Table 4.1 Genotypes and allele frequency of TDRD1 rs541192490 28 Table 4.2 Association between polymorphism TDRD1 rs541192490 and male infertility 29 vi LIST OF FIGURES Figure 2.1 piRNA biogenesis (Pek et al , 2012) 12 Figure 2.2 Structure of the typical eTudor domain (PDB: 3OMC) 13 Figure 2.3 Restriction enzyme cleavage for SNP genotyping (Kim & Misra, 2007) 16 Figure 3.1 PCR reaction condition 22 Figure 3.2 Recognition site of Psp1406I (AclI) 22 Figure 4.1 Total DNA of six samples in 1% agarose gel 26 Figure 4.2 PCR products amplified DNA region containing SNP TDRD1 rs541192490 on agarose gel 1% 27 Figure 4.3 Psp1406I (AclI) – digested PCR products of six samples on agarose gel 1.5% 27 Figure 4.4 Genotyping result of TDRD1 rs541192490 using Sanger sequencing 29 vii Part 1: INTRODUCTION In the recent decades, along with the population explosion, infertility has become a major health concern resulting in substantial psychological and social distresses (Bak et al., 2012) In Vietnam, a nationwide study of the National Hospital of Obstetrics and Gynecology and Hanoi Medical University also showed an ominous 7.7% of infertility rate in the childbearing age group Infertility affects 7% of the global male partner and about 40-50% of all infertility cases are due to the “male factor” (Sharlip et al., 2002; Dada et al., 2011; Krausz, 2011) The spermatogenic defect is the most common form of male infertility and idiopathic primary testicular dysfunction Many genetic factors are considered to contribute to this complex disease (Anawalt, 2013; Krausz et al., 2015) Spermatogenesis is a complex differentiation process of cells transformation from spermatogonia to mature spermatozoa, which is regulated by up to 2000 genes About 20% of these genes are found to be expressed only in male germlines (Schultz et al., 2003) Thus mutations or genetic alterations in these genes may lead to abnormalities in spermatogenesis resulting in male infertility (Stouffs et al., 2009) Piwi-interacting RNAs (piRNAs) are novel non-coding RNAs that play an essential role in spermatogenesis via piRNA biogenesis to protect the genomic DNA from transposon elements (Klattenhoff & Theurkauf, 2008; Yan, 2009) Numerous piRNA pathway-associated genes are found to be vital for the generation of piRNA (Olivieri et al., 2010; Saito et al., 2010) In particular, the Tudor domain-containing (TDRD1) gene is original from the Tudor gene encoding a protein that function in the suppression of transposable elements during spermatogenesis (Chen et al., 2009; Pillai & Chuma, 2012) It is located on the long arm of chromosome 10 at position 10q25.3, containing 30 exons The knockout model in mouse revealed the mutation in TDRD1 leading to complete male infertility due to spermatogenic defects (Chuma Lê Đình T Nguyễn Đình D Võ Văn T Lê Huy Q Lê Văn H Hoàng Văn N Trần Xuân D Đào Văn M Đào Đình P Dương Xuân N Lê Văn L Đoàn Văn T Bùi Văn L Bùi Ngọc T Vũ Thành L Hà Văn T Lê Anh V Nguyễn Sỹ H Vũ Văn S Nguyễn Minh T Bùi Thế H Nguyễn Hữu T Nguyễn Ngọc H Phạm Thanh T Nguyễn Văn T Trương Minh Đ Phạm Quốc A Lã Ngọc T Cao Văn T Nguyễn Văn Đ Nguyễn Đức A Nguyễn Tuyền 158 VSN225 P 159 VSN226 Đinh Đăng Đ 160 VSN227 Trần Văn T 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 VSN174 VSN175 VSN176 VSN179 VSN180 VSN181 VSN182 VSN184 VSN185 VSN186 VSN187 VSN188 VSN191 VSN192 VSN193 VSN194 VSN195 VSN197 VSN198 VSN199 VSN211 VSN215 VSN216 VSN217 VSN218 VSN219 VSN220 VSN221 VSN222 VSN223 VSN224 Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE 14 14.8 7.2 25.6 9.4 26.4 19.5 16.2 29.2 13.9 7.9 13.4 5.5 31.9 12.7 19.4 19.7 28.3 28.4 36.8 17.3 13.1 11.1 21 9.5 15.6 6.8 9.1 20.4 14.1 17.5 1.81 1.8 1.86 1.8 1.8 1.85 1.8 1.82 1.79 1.78 1.4 1.78 1.76 1.83 1.79 1.8 1.83 1.92 1.87 1.74 1.71 1.88 1.83 1.73 1.88 1.79 1.84 1.86 1.74 1.75 1.86 Male TRUE 23.2 1.7 Male Male TRUE TRUE 11 17.1 1.79 1.74 51 161 VSC008 Đào Mạnh Đ 162 VSC009 Phạm Văn T Nguyễn Mười 163 VSC010 Xuân A 164 VSC012 Ngô Văn L Nguyễn Thanh 165 VSC013 T 166 VSC014 Nguyễ Hữu V 167 VSC015 Diêm Công T Nguyễn Thành 168 VSC016 T 169 VSC017 Nguyễn Bá N 170 VSC018 Dương Hữu H 171 VSC019 Nguyễn Trung 172 VSC022 K 173 VSC025 Tạ Văn T 174 VSC026 Trần Thanh T 175 VSC027 Trần Ngọc A 176 VSC028 Nguyễn Xuân T 177 VSC029 Phạm Văn Đ 178 VSC031 Phạm Văn N 179 VSC033 Nguyễn Văn C 180 VSC034 Nguyễn Văn C 181 VSC035 Nguyễn Văn L 182 VSC036 Lê Văn M 183 VSC040 Hoàng Hữu H 184 VSC041 Hoàng Ngọc T 185 VSC043 Cấn Tiến T 186 VSC045 Trần Văn T 187 VSC048 Hồ Xuân H 188 VSC049 Nguyễn Đức T 189 VSC050 Trần Tuấn A 190 VSC051 Hà Văn C 191 VSC052 Đặng Đình K Male Male FALSE FALSE 17.1 21.4 1.72 1.79 Male FALSE 17.8 1.81 Male FALSE 19.5 1.74 Male FALSE 21.9 1.76 Male Male FALSE FALSE 20.4 14 1.69 1.76 Male FALSE 13.4 1.8 Male Male Male FALSE FALSE FALSE 14 13 11.8 1.72 1.74 1.73 Male FALSE 8.8 1.73 Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE 6.4 9.4 12.1 16.7 6.7 7.5 6.5 8.9 7.8 14.2 6.5 8.1 3.6 12.8 9.4 10.4 9.1 5.4 1.66 1.77 1.74 1.82 1.7 1.66 1.68 1.78 1.87 1.85 1.88 1.84 1.91 1.9 1.91 1.75 1.84 1.8 1.78 52 192 VSC053 Bế Tuấn H 193 VSC054 Lê Xuân Đ Nguyễn Mạnh 194 VSC055 T 195 VSC056 Trần Văn L 196 VSC057 Lê Xuân T 197 VSC058 Trần Việt A 198 VSC059 Vũ Xuân D 199 VSC060 Đinh Văn C 200 VSC061 Hoàng Văn N 201 VSC062 Phạm Công H Nguyễn Trung 202 VSC063 H 203 VSC064 Bùi Anh D 204 VSC065 Trần Quốc T 205 VSC066 Nguyễn Văn N 206 VSC067 Đỗ Việt T 207 VSC068 Phạm Văn H 208 VSC069 Nguyễn Anh D 209 VSC070 Trần Tiến T 210 VSC071 Đinh Xuân T 211 VSC072 Đặng Thanh T 212 VSC073 Trần Đức L 213 VSC074 Lê Việt C 214 VSC075 Tạ Nam S 215 VSC076 Trần Minh H 216 VSC077 Lê Viết H Nguyễn Quang 217 VSC078 T 218 VSC079 Nguyễn Hữu T 219 VSC080 Hoàng Văn T 220 VSC081 Lê Quang V 221 VSC082 Nguyễn Minh L 222 VSC083 Nguyễn Đình N 223 VSC084 Vũ Văn T Male Male FALSE FALSE 9.4 8.6 1.87 1.84 Male FALSE 11.3 1.9 Male Male Male Male Male Male Male FALSE FALSE FALSE FALSE FALSE FALSE FALSE 12.2 8.9 6.7 6.9 4.8 8.1 14.9 1.6 1.73 1.75 1.75 1.68 1.77 1.8 Male FALSE 10.6 1.77 Male Male Male Male Male Male Male Male Male Male Male Male Male Male FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE 9.9 9.8 3.1 6.2 10.5 10 1.2 6.3 7.8 12.9 11.6 4.5 13.4 10 1.69 1.82 1.5 1.53 1.78 1.81 4.35 1.68 1.7 1.78 1.7 1.68 1.73 1.76 Male FALSE 6.2 1.67 Male Male Male Male Male Male FALSE FALSE FALSE FALSE FALSE FALSE 6.8 4.8 5.5 5.8 7.1 6.9 1.61 1.66 1.65 1.68 1.68 1.78 53 224 225 226 227 228 229 VSC086 VSC087 VSC088 VSC089 VSC090 VSC091 230 VSC092 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 VSC095 VSC096 VSC097 VSC098 VSC099 VSC100 VSC101 VSC102 VSC103 VSC104 VSC105 VSC106 VSC107 VSC108 VSC110 VSC117 VSC119 VSC120 VSC121 VSC122 VSC123 VSC124 VSC125 VSC126 VSC127 VSC128 VSC129 Bùi Quang T Phùng Thế C Nguyễn Xuân T Hồ Viết B Nguyễn Đình Q Hồng Văn T Nguyễn Thành C Lưu Văn D Phạm Lê L Vũ Công N Lê Văn T Vũ Hồng C Nguyễn Đức V Lê Tuấn V Nguyễn Hải D Vũ Văn T Nguyễn Tuấn A Hà Văn T Vũ Văn H Trần Văn T Ngô Văn G Đào Văn H Vũ Văn H Vũ Văn T Lâm Văn H Hoàng Thành V Vũ Viết C Trần Hồng Q Phạm Văn H Vũ Văn T Đặng Ngọc T Hoàng Xuân Q Phạm Xuân Q Phạm Văn P Male Male Male Male Male Male FALSE FALSE FALSE FALSE FALSE FALSE 12.3 12.8 11.4 20.4 23.8 4.8 1.75 1.77 1.8 1.75 1.8 1.7 Male FALSE 5.1 1.77 Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE 7.2 6.7 7.1 10.5 21 13.2 15.4 11.3 25.5 15.3 19.6 11.2 7.7 14.6 25.9 13.4 22.1 15.6 24.4 12.5 25.1 19.8 16.8 15.6 15.1 18 1.72 1.83 1.7 1.53 1.78 1.57 1.63 1.72 1.78 1.75 1.73 1.84 2.05 1.76 1.7 1.77 1.78 1.68 1.78 1.72 1.73 1.57 1.5 1.48 1.63 1.65 1.68 54 258 VSC130 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 VSC131 VSC132 VSC133 VSC134 VSC135 VSC136 VSC137 VSC138 VSC139 VSC140 VSC141 VSC142 VSC143 VSC144 VSC146 VSC147 VSC148 276 VSC149 277 278 279 280 281 282 283 284 285 286 VSC150 VSC154 VSC155 VSC156 VSC158 VSC159 VSC160 VSC161 VSC162 VSC164 287 VSC165 288 VSC166 289 VSC167 Nguyễn Tuấn M Nguyễn Văn K Nguyễn Như T Lưu Ngọc K Vũ Văn D Phạm Văn S Nguyễn Ngọc S Nguyễn Công Đ Lê Quang H Nguyễn Hồng T Đào Văn H Trần Văn H Đặng Đình Đ Nguyễn Tiến Đ Lương Thế H Vy Văn H Phạm Văn P Trần Quốc H Nguyễn Công Tuấn A Phạm Xuân C Lê Văn L Trần Thái K Đặng Việt S Đỗ Xuân H Bùi Duy C Phan Công T Vũ H Lê Văn B Phạm Văn Đ Nguyễn Thành L Nguyễn Vĩnh B Phan Quốc P Male FALSE 24.6 1.76 Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE 18.6 14.1 16.9 25.9 15.3 11.6 6.1 10.4 6.2 14 12.1 8.8 13.1 12.7 16.2 35.6 10.4 1.72 1.76 1.83 1.69 1.72 1.71 1.65 1.79 1.73 1.86 1.88 1.78 1.74 1.84 1.79 1.82 1.6 Male FALSE 14.2 1.66 Male Male Male Male Male Male Male Male Male Male FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE 14.7 37.9 13.3 24 10.5 4.8 4.9 12.8 8.8 12.4 1.81 1.82 1.9 1.86 1.84 1.83 1.89 1.75 1.85 1.74 Male FALSE 11.5 1.83 Male Male FALSE FALSE 11.2 13.7 1.91 1.72 55 290 VSC168 291 292 293 294 295 296 297 298 299 300 301 VSC169 VSC170 VSC171 VSC172 VSC173 VSC174 VSC175 VSC176 VSC178 VSC179 VSC180 302 VSC181 303 VSC182 304 VSC183 305 VSC184 306 VSC185 307 308 309 310 VSC186 VSC187 VSC188 VSC190 Nguyễn Phùng D Phạm Văn N Đoàn Trung L Long Minh H Quách Ngọc H Nguyễn Văn H Trịnh Xuân T Dương Ngô T Nguyễn Văn H Trần Minh T Đào Văn T Nguyễn Viết A Nguyễn Hoàng H Nguyễn Văn T Lê Văn L Trịnh Viết H Nguyễn Trường D Ngơ Thế T Trần Đình T Đình Đức D Quàng Mạnh H Male FALSE 27.7 1.83 Male Male Male Male Male Male Male Male Male Male Male FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE 15 34.7 26.7 13.1 12.6 3.8 6.9 41.9 6.3 11.8 6.5 1.75 1.81 1.83 1.83 1.81 1.68 1.96 1.74 1.66 1.68 1.85 Male FALSE 13.8 1.71 Male Male Male FALSE FALSE FALSE 13.4 33 15.2 1.84 1.81 1.76 Male FALSE 29.3 1.76 Male Male Male Male FALSE FALSE FALSE FALSE 18.4 19.7 13.4 24.1 1.8 1.94 1.79 1.73 56 Appendix DNA extraction result 57 Figure Electrophoresis of total DNA extraction of 310 samples 001 – 154: Patient VSN012 to VSN227; 155 – 310: Control VSC008 to VSC190 58 Appendix 3: PCR result 59 Figure Electrophoresis of PCR products of DNA region containing TDRD1 rs541192490 of 310 samples 60 M: Marker 100 bp; P: Positive control; N: Negative control 001 – 154: Patient VSN012 to VSN227; 155 – 310: Control VSC008 to VSC190 61 Appendix PCR-RFLP result 62 Figure Electrophoresis of Psp1406I – digested PCR products of 310 samples M: Marker 100 bp; UC: uncut product 001 – 154: Patient VSN012 to VSN227; 155 – 310: Control VSC008 to VSC190 63 Appendix Sanger sequencing result (2) (1) VSN057 VSN014 (3) (4) VSN150 VSN181 (6) (5) VSN046 VSN025 (7) (8) VSN164 (9) VSN118 (10) VSC005 VSC031 (12) (11) VSC098 VSC035 64 (13) (14) VSC055 VSC146 (15) VSC180 Figure Sanger sequencing result of 15 samples 1-8: Patients with wildtype form GG 9-15: Controls with wildtype form GG 65