Identification of pathogenic variants in IGHMBP2 gene and application on prenatal diagnosis of rare neuromuscular disorders

CAO HA MY IDENTIFICATION OF PATHOGENIC VARIANTS IN IGHMBP2 GENE AND APPLICATION ON PRENATAL DIAGNOSIS OF RARE NEUROMUSCULAR DISORDERS Graduation Thesis Major: General Practitioner Major code; 52720101 Aradrmir Super hor Associate Professor TRAN VAN KHANH M.D PhD I «s> ■> ACKNOWLEDGEMENT first joined scientific research four years ago as an enthusiastic student yearning for new knowledge That full of difficulties but memorable experiences have changed me and become part of my youth I was lucky enough to meet, learn, and receive valuable guidance from many teachers and friends during my journey Their support would be of great importance for me to complete my graduation thesis confidently Firstly would like to express my deepest gratitude to Associate Professor Tian Van Khanh M D Ph D , 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 thesis successfully am thankful to her for presenting such excellent advice and guidance despite having a tight schedule I heartily thank Professor Ta Thanh Van MD, PhD Chairman of University Council of Hanoi Medical University and Director of the Center for Gen Protein Research, Professor The Hung Bui, MD, PhD Centre for Molecular Medicine Clinical Genetics Unit in Karohnska Universitat, and Assc Professor Tran Huy Thinh MD PhD Deputy 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 Luong Hoang Long Department of Clinical Allergy Immunology and Dermatology National E hospital for his patience consistent motivation and immense knowledge He also always provided me with insightful comments and hard questions during my scientific research projects -w ã* CN ôG My sincere thank also goes to Ms Le Thi Phuong and the Centre for GeneProtein Research staff who carefully instructed and supervised me to conduct several molecular techniques The last words of thanks I would like to send to my parents, who are the idols io me and have always laid concrete encouragement to me in my life Hanoi May 2021 Cao Ha My -w ã* CN ôG DECLARATION 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 th? text Hanoi May 2021 CAO HA MY -w ã* CN ôG LIST OF ABBREMATIOX AchR Acetylcholine receptor AFP Alpha fetoprotein CNS Central nervous system CMT2S Charcot-Marie-Tooth disease type 2S CVS Chorionic villus sampling D1A Dimeric Inhibin-A DNA Deoxyribonucleic Acid DSMA Distal Spinal Muscular Atrophy hCG human Chorionic Gonadotropin HMN Hereditary Motor Neuropathy HMSN Hereditary motor and sensory neuropathies 1GHMBP Immunoglobulin Mu DNA Binding Protem IVF in \ìtrữ Fertilization NMD Neuromuscular disorder NGS Next Generation Sequencing PNS Peripheral nervous system Papp-A Pregnancy associated plasma protein A PGĐ Preimplantation Genetic Diagnosis PGS Pre implantation Genetic Screening PCR Polymer Chain Reactions RNA Ribonucleic Acid SMARD1 Spinal Muscular Atrophy with Respiratory distress S.MA Spinal Muscular Atrophy nE3 L'nconjugated Estriol -w •* CN «G TABU, of contents INTROD VCTION .—MMMM CRAFTER LITERATURE REVIEW 1.1 Phenotypic spectrum of Neuromuscular Disorders 1.1 The peripheral nervous systems ••••»•• 1 Classification of Neuromuscular disorders and Congenital Neuromuscular disorders •• 1 Spinal Muscular Atrophy with Respiratory distress 1 Charcot-Marie-Tooth disease type’s 13 1.2 The history of IGHMBP2 gene 12.1 Gene structure and mapping 12.2 Gene function 123 Molecular genetics 19 2Ơ » 1.3 Prenatal Screening and Prenatal Diagnosis in Genetic diseases 24 I Difference between prenatal screening and prenatal diagnosis 24 132 Prenatal screening 133 Prenatal diagnosis — CHAPTER SUBJECTS AND METHODS- 29 2.1 Time and location 29 22 Subjects 29 2.3 Laboratory’equipment and apparatus I Laboratory equipment 23.2 Chemicals .29 29 30 2.4.1 Research protocol - - - 31 2.4.2 DN A extraction — 32 2.4 Pathogenic variants identification using Sequencing methods 34 44 Prenatal diagnosis protocol 2.5 Study ethics 35 36 CHAPTE R R ESI LTS 1« 3.1 Identification ơfIGHMBP2 pathogenic variants and clinical manifestation of the patients 3.1 Clinical manifestation of four patients 37 3.1-2 Identification of ỈGHMBP2 pathogenic variants 41 3.2 Identification of carriers among family members and application on prenatal diagnosis — — 41 CHAPTER DISCUSSION _ 45 4.1 Clinical manifestations and genetic testing of patients and family members 45 4.2 Pathogenicity classification QĨÌGHMBP2 variants 4.3 Options for prenatal diagnosis for hereditary disease CHAPTER CONCLUSION RE FERE NCE APPEND LX -w -r? M «G 51 55 LIST OF FIGURES Figure 1.1 Venn diagram of disease genes for Charcot-Marie-Tooth disease Figure Location of ỈGHMBP2 gene on chromosome 11 19 Figure 1.3 Structural overview of human Ighmbp2 protein and the form of human lghmbp2 that combining with an RN*A .9 20 Figure I IGHMBP2 genes and pathogenic variants 22 Figure 2.1 Research protocol Figure 2 Prenatal diagnosis protocol Figure Clinical manifestations of patient and patient 39 Figure Patient family pedigree and genotype 42 Figure 3 Patient ốmily pedigree and genotype 42 Figure Patient family pedigree and genotype 43 Figure s Patient family pedigree and genotype Figure Family pedidree and genotype after prenatal diagnosis and genetic 31 35 43 counselling 44 Figure Simplified IGHMBP2 protein model 52 Figure 4.2 Protein model simulation of IGHMBP2 protein 52 Figure Location of C1574TX? (pLeu525Pro) on 1GHMBP2 protein simulating model - Figure 4 Rules for combining criteria to classify sequence variants -w ã* CN ôG - 53 55 LIST or TABLES List of genes identified as distal hereditary motor neuropathies (dHMN) Table 1.1 based on the original classification by Harding and Thomas1980 10 T able -2 Classification of Axonal CMT according to genotype 18 Table J Overview of current serum screening options for aneuploidv 26 Table 1ỈGHMBP2 primers Table Comparison of clinical characteristics and IGHMBP2 variants 39 Table Information of IGHMBP2 variants found in patients 44 Table 1SMART)1 diagnostic criteria (from Pitt et al.) -w ã* CN ôG 35 ãằ* 48 Identification of Parhogenic Variants in ICHMBPỈ gene and Application on Prenatal Diagnosis of Rare Neuromuscular Disorders ABSTR ACT Background Spinal muscular atrophy with respiratory distress type (SMARD1) and Charcot - Marie - Tooth npe 2S (CMT2S) arc rare neuromuscular disorders caused tn- biallelic pathogenic variants on IGH MBP2 Objectives: 1) To identify pathogenic variants in ỈGHMBP2 gene and describe four cases with IGHMBP2 mutation 2) To identify carriers and application on prenatal diagnosis Subjects and methods: Four patients under 12 years of age with lower limbs weakness (3 had respirators- disorders) and family members Genetics analysis for patients and family members was performed using Next Generation Sequencing and Sanger Sequencing Results We identified four IGHMBP2 mutations, in which c 1574T> c (p Leu525Pro) is a novel mutation Both parents and sisters of four patients were idem I tied 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 ỈGHMBP2 Conclusion: patient was homozygous for ÍŨHMBP2 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 respiratory distress Charcot-Marie-Tooth SMARD1 neuromuscular disorder, 53 Based oa international mutatxxi databases such as Human Gene Mutation Database LOV'D - Human Variome Project ClmVar - NCBI we confirmed el5"4T=€ (p.Leu525Pro> to be a novel valiant The pathogenicity of each IGHM3P2 variant was carefully accessed with the support of computational modeling programs, literature review and American College of Medical Genetics (ACMG) guidelines for classification of variant pathogenicity [51] In sihcữ programs used were Mutation Taster FAT HMM and DANN [60], [61], [62] (detail in Table 2) These computation programs all classified die novel variant c 1574T>C (p Leu525Pro) to be pathogenic - Mutation taster returned the results of "Disease causing'* - FATliMM consider the variant to be “Damaging" - DANN scored the variant with “0.9989/1 *■ Using Vniprot and Swissmodel we were able to discover that the amino acid change resided in the alpha-helix (Figure 3) [63] [64] The original amino acid was leucine which helps straighten and keep die alpha helix in proper form Cordes et al (2002) explained the difference of alpha-helix with LeucÍDe versus Proline since proline cannot produce hydrogen bond it leads to the alpha-helix hinge (bending) [65] This could be the possible mechanism for protein function loss regarding c 1574T>C (p Leu525Pro) variants Ffgtrre 4.3 Location of C.15~4T>C ịp.Leu525Proị on ỈGHMBP2protein simulating model (I'niprol Accession number PỈS9ỈĨ) 1- CN «G -4: 54 The ACMG guidelines for classification of variant pathogenicity was applied for further accuracy which returned with five appropriate criteria (DPMI: C.1574T>C (pLeu525Pro) is located in die helicase domain of the ỈGHMBP2 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 Tabk 3.2) (3}PM3 patient had compound heterozygous for two ĨGHMBP2 variants cl235-3A>0 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 vtĩro evidence (Guenther 2007), the variant C.1574TX? (p.Leu525Pro) is trans with a pathogenic variant thereby fulfilling the requirement of PM-' Noted that these criteria are only accepted for recessive disorders (4) pp Multiple lines of computational evidence support a deleterious effect on the variant (as shown in Table 3.2) (5JPP5- Lniprot 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 perform an independent evaluation These criteria allow US to classify C.1574T>C (pLcu525Pro) to be "likely pathogenic’* according to ACMG guidelines (f igure 4.4 -w ã* CN ôG 55 ijfcM ằ vartantl '■> »ow>b>»v«9 I Ui** »V1 IV1Í > anỉ *0ô ô*>0* ã* rlto-rtn)*! HS1 R -ằ$*>Êằ MlSacrvlKI-KOMO uMUWikW ằMằOằ rM&MOa W"ằg'ằl M$|ô |r)l IA.X.W.PMI 4T4 >> **ằãã MMằVfcOằ « t ViKryiMt »4> AO i-,mjooij«» ỊVI|Ũ(>C* ■• lfeo«Ml KA’MMOaiMVoan* OI Mt>0* 44 r>M>ằ^.to.ôM- MJftoa ã* JMjdxtO J*lt PM6! AO.J'.CTMUx; M M4,.*w^v< ôx ASO.l -A^-ô-ớ 01 Ob ãmq* a >Wằằ*rằw**lOr IMiMnan ã aKVQKI rx ô >èMneếl-*M>ôôllô4Mtae ằW) iranằ H)|LJ*" 4* OMvoMmvxwxnaMMMmoa « tw«9» Mt N”-*~ «M 4W**«W|r _ Figure 4.4 Rules for comờ fifing criteria ro classify sequence variants (reproducing from J CMG guidelines for variant pathogen Icky - 2015) Ị66Ị 43 Options for prenatal diagnosis for hereditary disease Genetic analysis divulged that parents of four patients are IGHXÍBP2 carriers Since SMARD1 and CMT2S Me autosomal recessive disorders the probability of carrier parents having an infected child is 25% Thus, genetic courseling such as prenatal diagnosis or pre-implant diagnosis would allow parents to lower the possibility of having a homozygous child At the time of the research, the mother of patient family was 17-week- pregnant Since patient three was diagnosed with SMARD1 with compound heterozygous variants, the family was advised to conduct amniocentesis as genetic diagnostic testing Genetic analysis of the fetus revealed a compound heterozygous TWM*M«K> «“ *4: 5Ổ genotype suggesting that the fetus would suffer from SXCARDi if he she was bom After thorough genetic counseling the parents decided to terminate the pregnancy Prenatal genetic diagnostic testing IS intended to determine whether a specific genetic disorder or condition is present in the fetus with as much certainty as possible Prenatal genetic testing has many benefits, including reassuring patients when results are typical identifying disordas for which prenatal treatment may benefit, optimizing neonatal outcomes by ensuring the appropriate location for delivery' and the necessary personnel to care for affected infants, and allowing the opportunity for pregnancy termination As mentioned in chapter chorionic villus sampling (CVS), arnniocentesữ and preimplantation genetic diagnosis (PGD) are currently the practical tools for intra uterine genetic diagnosis Chorionic villus sampling for prenatal genetic diagnosis is generally performed between 10 weeks and 13 weeks of gestation, while amniocentesis is for gestational age after 15 weeks The primary advantage of CVS over amniocentesis is that the procedure can be performed earlier in pregnancy and the viable cells obtained by CVS for analysis allow for shorter specimen processing time (5 day's versus H days) so the results are available earlier in pregnancy After an abnormal first-trimester ultrasound examination or screening lest, the earlier CVS results allow for more management options although amniocentesis also is an option for diagnosis [48] The presjiancy loss rate from CVS has decreased over time The most recent meta analysis of studies that included a control group including 899 women with CVS and 37.388 who had no procedure, calculated a procedure-related loss rate of 0.22% (1 in 455) [Akolekar 2015 Bulletin 162] Although there have been reports of an association between CVS and lirub reduction defects the risk of these anomalies appears to be very’ low, and the anomalies are more Significant with procedures performed earlier than ten weeks of gestation [67], Another complication of CVS is vaginal spotting or bleeding which may occur in up to 32% of patients after transcervical CVS [68] the incidence alter trans abdominal CVS is -w ã* CN ôG 57 lower than that The incidence of culture failure amniotic fluid leakage or infection after CVS is less than 5% (68) (69) Amniocentesis for genetic diagnosis usually is performed between 15 weeks and 20 weeks of gestation but it can be performed at any later gestational age Manx* extensive multicenter studies have confirmed the safety of genetic amniocentesis and its cytogenetic diagnostic accuracy (Jackson NEJM) If technically feasible, transplacental passage of the needle usually is avoided, especially in cases invoking alloimmunừation although the data suggest that the procedure-related loss rate is not different with transplacental and nontransplacental approaches (70) (71) The procedure often is postponed if the amnion and chorion have not been fused because there is a higher likelihood of failing to obtain amniotic fluid or requiring a second puncture The most significant risk of amniocentesis is pregnancy loss As with CVS the procedure-related loss rate of mid-trimester amniocentesis has decreased over time likely because of increasing experience and improvements in technique and imaging Accurate data on miscarriage after amniocentesis are challenging to obtain because of the rarity of the outcome and the difficulty in comparing women who experience miscarriage after amniocentesis with an appropriate control group The rate of procedure-related pregnancy loss attributable to a prenatal diagnostic procedure is estimated to be approximately 3% in procedures performed by experienced healthcare providers However, there are different findings depend on the medical setting and physician's technique Also, when counseling patients about the possibility of miscarriage after amniocentesis it is crucial to place the procedure-related risk in the context of the patient's background risk Minor complications from amniocentesis occur infrequently and include transient vaginal Spotting or amniotic fluid leakage 111 approximately 2% of all cases (72) In the past early amniocentesis has been performed between 10 weeks and 13 weeks of gestation using a technique similar to mid-ưimester amniocentesis [73] However, early amniocentesis has significantly higher rates of pregnancy loss and other -w CN «G 58 complications than mid trimester amniocentesis Therefore early amniocentesis (before 14 weeks of gestation) is not recommended Preimplantation genetic draptosw refers to the testing of an embryo for a specific genetic disorder before implantation Pre implantation genetic testing is performed on polar bodies from the oocyte and zygote a single blastomere from a cleavage-stage embryo, or a group of cells from the trophectoderm at the blastocyst Stage A preunplantation genetic diagnosis can be performed using cytogenetic or molecular techniques on early embrvos created by in vt.Tữ fertilization and can be used to test for most genetic conditions in which a mutation has been identified in the family Because preimplantaãon genetic diagnosis uses only one or a few cells from the early embryo and errors are possible, confirmation of results with CVS or amniocentesis is usually recommended -w ã* CN ôG 59 CHAPTER CONCLUSION In this research we reached two conclusions Firstly 14 patient was homozygous for an ỈGHMBP2 variant and had compound heterozygous variants Four variants found included: C.1235-3AX3 (intron 8) c 1334A>C (p Hts445Pro) (exon 9) C.1574T>C (pLeu525Pro) (exon 11) C2362OT (p Arg788Ter>(cxonl3), in which C.1574TX? (pLeu525Pro) is a novel variant All patients presented with lower limbs muscle weakness Only patients developed respiratory distress, and two of them deceased before the age of two Secondly, parents and siblings of four patients were all earners and genetic testing was successfully applied on prenatal diagnosis for the family of patient -w •* CN «G REFERENCE 2_ Hubbard J ed (1974), 77» Peripheral Nervous System springer US < 1078) A clinician** view of neuromuscular diseases, by Michael II Brooke 225 pp illustrated $18 95 the ^ĩllianis & Wilkins Company, Baltimore 197' , el08-122 49 Akolekar R, Beta J, Picciardli G , et al (2015) Procedure-related risk of miscarriage following amniocentesis and chorionic villus sampling a systematic review and meta-analysis Ultrasound Oste Gynecol 45(1) 16 26 -w ã* CN ôG 50 Hirton GL De Rỵcke M Fiorentino F et al (2011) ESHRF PGD consortium best practice guidelines for amplification-based PGD HumReprod 26(1) 33 40 51 Rich aids s, Aziz N, Bale s, et al (2015) Standnds and guidelines fox the interpretation of sequence variants: a joint consensus xecommendarion of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology Generics Pi Xiedictne 17(5), 405-423 52 Ircbi J (2004) Molecular genetics of distal hereditary motor neuropathies Human Molecular Genetics 13(suppl_2) R.195 R202 53 Guenther u p VtfOA R Schlicke M « al (2007) Clinical and mutational profile in spcnal muscular atrophy with respiratory distress (SMARD) defining novel phenotypes through cluster hierarchical analysis Human '-https onlinelibrary Wiley com doi kbs 10 1002 humu 2O525> Mutation, accessed OS 14/2019 $4 Jfdrzcjowska M Madej-Pihuvzyk A FidziaAska A Ct al (2014) Severe phenotypes of SHARD I associated with novel mutations of the ÌGHMBP2 gene and nudear degeneration of muscle and Schwann cells European Journal of Paediatric Neurology 18(2) 183 192 55 Pcdurujillay c R J , Amundsen s s , Barov T , et al (20Ỉ6) Clinical and molecular characteristics in three families with biallelic mutations in IGHMBP2 Vraownttfeulp* D/Mn/enr, 2fi(9), 570 575 56 Luaa X Huang X Liu X et al (2016) Infantile spinal muscular atrophy with respiratory distress type preventing without respữatory involvement; Novd mutations and review of the literature Bra'.r and Daveỉopmanĩ, 33(7) 6S5-6S9 5" Ikeda A Yamashita s Tsuyusaki Y et al (201S) Peripheral nerve pathology at fixed stage in spinal muscular atrophy withrespiratory distress type Bra'.n and Daveiopfueni 40t 2) 155-158 58 Saladini M Nizzardo M Govoni A et al (2020) Spinal muscular atrophy with respirators- distress tvpe I Clinical phenotypes, molecular pathogenesis and therapeutic insights Journal of Cellular and Molecular Međicino 24(2), 1169 1178 59 Tomaselli PJ , Horga A Rossor A M et al (2018) IGHMBP2 mutation associated with organ-specific autonomic dssfunction Xđuromuscul Disord, 28(12), 1012 1015 -w •* CN «G e 6Õ Schwarz ĩ \ỉ , Cooper D N Schueỉke M et al (2014) \íuta:;ũnTaíter2 mutation psediơion for the deep-Vequenciag age \'or Methods, 11(4) 361 362 61 Rog«s MF Shihab HA, Mon M « al