Identification of Pathogenic variants in IGHMBP2 gene and Application on Prenatal diagnosis of Rare Neuromuscular Disorders

CAO HA MY IDENTIFICATION OF PATHOGENIC VARIAN TS IN IGHMBP2 GENE AND APPLICATION ON PRENATAL DLAGNOSIS OF RARE NEUROMUSCULAR DISORDERS Graduation Thesis Major: General Practitioner Major code: 52720101 Academic Supervisor Associate Professor TRAN VAN KHANH M.D, PhD ACKNOWLEDGEMENT I first joined scientific research four years ago as an enthusiastic student yearning for new knowledge That full of difficulties but memorable experiences have -■c -ÍM V changed me and become part of myQỉ ugc youth I was lucks' enough to meet, learn, and Hl receive valuable guidance from many teachers and friends during niv journey Their support would be of great importance for me to complete my graduation thesis confidently Firstly I would like to express my deepest gratitude to Associate Professor Tran Van Khanh MD PhD Deputy Director of Center for Gene-Protein Research Head of Molecular Pathology Department of Hanoi Medical University, who provided me great support and leadership which allowed me to conduct the research and finish this diesis successfully I am thankful to her for presenting such excellent advice and guidance despite having a tight schedule I heartily thank Professor Ta Thanh Van, M.D, Ph D Chairman of University Council of Hanoi Medical University and Director of die Center for Gen-Protein Research Professor The-Hung Bui MD, PhD Centre for Molecular Medicine Clinical Genetics Unit in Karolinska Universitat, and Assc Professor Tran Huy Thinh M.D., Ph D Deput)' Head of Department of Biochemistry Head of Department of Technology and Scientific Research Management, who always created favorable conditions for me to access scientific research and inspired me to be a good doctor and researcher with a thorough understanding of medical ethics I am deeply thankful to Dr always Luong Hoang Longme Department Clinical hospital Allergy for his patience, Immunology consistent and Dermatology motivation National and Eof immense insightful knowledge comments He and also hard questions provided during with my scientific research projects -ÍM Qỉ ugc V Hl My sincere thank also goes to Ms Le Thi Phuong and the Centre tor GeneProtein Research staff who carefully insttucted and supervised me to conduct several molecular techniques The last words of thanks would like to send to my parents, who are the idols in me and have always laid concrete encouragement to me in my life Hanoi May 2021 Cao Ha My r-u -ÍM CỊỈ ugc V Hl DECLARATION LIST OF ABBREVIATION I hereby certify that this thesis incorporates original research, which has not been previously submitted for a degree to any other institution, and the best of my knowledge and belief, it does not contain any material previously published or written by other persons except where reference has been made in the text Hanoi May 2021 CAO HA MY AchR AFP Acetylcholine receptor Alpha-fetoprotein CNS Central nervous system CMT2S CVS Charcot-Marie-Tooth disease, type 2S Chorionic villus sampling DIA Dimeric Inhibin-A DNA Deoxyribonucleic Acid DSMA hCG Distal Spinal Muscular Atrophy human Chorionic Gonadotropin HMN Hereditary Motor Neuropathy HMSN IGHMBP Hereditary motor and sensory neuropathies Immunoglobulin Mu DNA Binding Protein IVF in vứro Fertilization NMD Neuromuscular disorder NGS PNS Next Generation Sequencing Peripheral nervous system Papp-A Pregnancy-associated plasma protein A PGD Preimplantation Genetic Diagnosis r-u -ÍM Qỉ ugc V Hl PGS Preimplantation Screening LIST Genetic OF ABBREVIATION PCR Polymer Chain Reactions RNA Ribonucleic Acid SMARD1 SMA Spinal Muscular Atrophy with Respiratory distress Spinal Muscular Atrophy uE3 Unconjugated Estriol r-u -ÍM Qỉ ugc V Hl TABLE OF CONTENTS INTRODUCTION REFERE NCE APPEND LX -■c -ÍM Qỉ ugc V Hl LIST OF FIGURES LIST OF T ABLES Table 1.1 List of genes identified as distal hereditary motor neuropathies (dHMN) Identification of Pathogenic Valiants in IGHMBP2 gene and Application on Prenatal Diagnosis of Rare Neuromuscular Disorders ABSTRACT Background spinal muscular atrophy with respiratory distress type (SMARD 1) and Charcot - Marie - Tooth type 2S (CMT2S) are rare neuromuscular disorders caused by biallelic pathogenic variants on IGHMBP2 Objectives: 1) To identify pathogenic variants in IGHMBP2 gene and describe four cases with IGHMBP2 mutation 2) To identify’ earners and application on prenatal diagnosis Subjects and methods: Four patients under 12 years of age with lower limbs weakness (3 had respiratory disorders) and family members Genetics analysis for patients and family members was performed using Next Generation Sequencing and Sanger Sequencing Results We identified four IGHXỈBP2 mutations, in which C.1574T> c (p Leu525Pro) is a novel mutation Both parents and sisters of four patients were identified to be carriers The mother of the third patient’s family was pregnant but had an abortion after genetics testing of the fetus revealed compound heterozygous mutations on IGHMBP2 Conclusion: patient was homozygous for IGH.\iBP2 mutation, and three out of patients had compound heterozygous mutations All parents were identified as carriers, and we successfully applied the genetic testings on prenatal diagnosis for the family of patient 03 Key words: IGHMBP2, muscle weakness neuromuscular respiratory distress Charcot-Marie-Tooth SMARD1 disorder INTRODUCTION Neuromuscular disorders (NMDs) include various conditions that affect components of a motor unit, sensor.' and autonomic nen es, or their supportive r-u -ÍM CỊỈ ugc V Hl 68 accuracy, which returned with five appropriate criteria (1) PMl: C.1574T>C (pLeu525Pro) is located in the helicase domain of the IGHXÍBP2 gene, which also the well-established functional domain of the protein (2) PM2: the variant was not found in the 1000 Genome Project of the South Asian population 9as shown in Table 3.2) (3) PM3: patient had compound heterozygous for two ỈGHMBP2 variants c 1235-3A>G and C.1574T>C (p Leu525Pro) Mom was a carrier with the former, indicating that the latter for received from her father Since the former was previously found to be pathogenic and had in vitro evidence (Guenther 2007) the variant C.1574T>C (p.Leu525Pro) is trans with a pathogenic variant, thereby fulfilling the requirement of PM3 Noted that these criteria are only accepted for recessive disorders (4) PP3:Multiple lines of computational evidence support a deleterious effect on the variant (as shown in Table 3.2) (5) PP5: Uniprot simulated that the variant could lead to an alpha helix change, causing protein function loss However, due to our lack of laboratory equipment we could not -c -ÍM Qỉ ugc V Hl H& 69 perform an independent evaluation These criteria allow US to classify C.I574T>C (p.Lcu525Pro) to be “likely pathogenic" according to ACMG guidelines (figure 4.4) -c -ÍM Qỉ ugc V Hl H& 70 ToMo Ỉ Rul« for combining criteria to (Unify MKjucrKC varunti p*"«>xoc C (p His445Pro) (exon 9) C.1574T>C (p.Leu525Pro) (exon 11) C.2362OT (p Arg788Ter) (exonl3), in which C.1574T>C 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