Xác Định Đột Biến Gen Gla, Gaa Và Đặc Điểm Di Truyền Của Bệnh Fabry Và Pompe Ttta.pdf

MINISTRY OF EDUCATION MINISTRY OF HEATH AND TRAINING HANOI MEDICAL UNIVERSITY NGUYEN THI PHUONG THAO DETECTION OF GLA, AND GAA GENE MUTATIONS AND THE GENETIC CHARACTERISTICS OF FABRY AND POMPE DISEASE Specialism : Biochemistry Code : 9720101 ABSTRACT OF THESIS HA NOI - 2023 The thesis has been completed at HANOI MEDICAL UNIVERSITY Supervisors: Supervisor 1: Prof Ta Thanh Van M.D., Ph.D Supervisor 2: Assoc Hoang Thi Ngoc Lan M.D., Ph.D Reviewer 1: Reviewer 2: Reviewer 3: The thesis will be presented in front of the board of university examiners and reviewer lever at… on ….20 This thesis can be found at: National Library: National Medical Informatics Library Library of Hanoi Medical University THE LIST OF WORKS PUBLISHED AND RELATED TO THE THESIS Nguyen Thi Phuong Thao, Nguyen Thi Phuong Mai, Dau Ming Niu, Nguyen Van Hung, Hoang Thi Ngoc Lan, Ta Thanh Van (2019) Detection of GLA gene mutation in a family with Fabry disease in Vietnam, Vietnam medical journal No Đặc biệt (Vol 484), 645 -650 Nguyen Thi Phuong Thao, Dau Ming Niu, Nguyen Quynh Tho, Hoang Thi Ngoc Lan, Ta Thanh Van (2020) Prenatal diagnosis for pregnant women with GAA gene mutation causing Pompe disease, Vietnam medical journal No Chuyên đề (Vol 496), 205 – 210 Nguyen Thi Phuong Thao, Vu Chi Dung, Nguyen Ngoc Khanh, Le Thi Phuong, Tran Van Khanh, Hoang Thi Ngoc Lan, Ta Thanh Van (2023) Detection of GAA gene mutation and inherited trait of Pompe disease, Journal of medical research Hanoi Medical University No (Vol 164), 18 – 24 INTRODUCTION Fabry and Pompe are two rare genetic diseases belonging to the group of lysosomal storage disorders - LSDs The cause of the disease was determined to be a mutation in GLA or GAA genes, leading to a deficiency of the lysosomal enzyme alpha-galactosidase A or alpha-glucosidase, resulting in the accumulation of sphingolipids, glycogen, respectively, in the lysosome, Although the incidence is very low, children with these diseases often die early due to heart failure and respiratory failure Recent studies report a nonsmall proportion of atypical Fabry cases The detection of disease-causing gene mutations and the detection of healthy people carrying disease genes are of great significance in early diagnosis, treatment, and reasonable genetic counseling to limit the birth of sick children However, in Vietnam, there are very few studies on these diseases, leading to late diagnosis and low treatment effectiveness For the above reasons, we conducted the thesis “Detection of GLA and GAA gene mutations and genetic characteristics of Fabry and Pompe disease” with three following objectives: Identifying mutations in GLA and GAA genes in patients with Fabry and Pompe disease Detect carriers on family members of patients Describe the main clinical and paraclinical manifestations of patients with Fabry and Pompe disease Necessity of the thesis Fabry and Pompe are two rare genetic diseases belonging to the group of lysosomal storage disorders - LSDs Although the incidence is low, children with the disease often die early due to heart failure and respiratory failure Recent studies report a small proportion of atypical Fabry cases The detection of disease-causing gene mutations and the detection of healthy people carrying disease genes are of great significance in early diagnosis, treatment, and reasonable genetic counseling to limit the birth of sick children These are the reasons why our study was conducted New contributions from the thesis - Identification of GLA, and GAA gene mutations causing Fabry and Pompe disease in Vietnam, including 01 new GLA gene mutation and 02 GAA gene mutation - Detecting carriers on family members with Fabry, and Pompe disease, thereby providing appropriate genetic counseling for each subject - Provide data on clinical characteristics (common symptoms) and paraclinical changes (biochemistry, imaging) of patients with Fabry and Pompe Thesis outline The thesis consists of 128 pages, covering: the introduction (2 pages), overview (37 pages), object and method of research (20 pages), results (36 pages), discussion (31 pages), conclusion (1 page), proposal (1 page) It has 18 tables, 20 figures, and charts 155 References Chapter OVERVIEW 1.1 Fabry disease – FD - Fabry is an X-linked genetic disease caused by excessive accumulation of sphingolipids in the lysosome, leading to various organ symptoms, as a consequence of alpha-galactosidase A deficiency (GLA), caused by mutations in the GLA gene, which encodes the production of the GLA enzyme - The GLA gene is located on the long arm of the X chromosome, at position Xq22.1 The gene consists of exons and about 10,222 bp The coding region is 1290 bp, encoding a protein of 429 amino acids To date, more than 900 mutations in the GLA gene causing Fabry disease have been reported - Fabry is a rare disease, with an incidence ranging from 1/40,0001/60,000 people - Clinical manifestations of Fabry are varied with manifestations of pain, corneal or skin lesions, and eventual organ failure, common in children aged 3-10 years, in girls usually later in boys The mechanism of tissue injury is thought to be hypoperfusion due to the accumulation of substances in vascular epithelial cells alone, particularly in the kidney, heart, central nervous system, and skin, or in combination with the accumulation of vascular epithelial cells deposited in other tissues The heterozygous female may or may not be symptomatic, due to the random inactivation of the X chromosome in all cells of the female embryo The disease can be diagnosed based on clinical symptoms, GLA enzyme activity tests, or genetic analysis tests for mutations - Currently, enzyme replacement therapy (ERT) is considered the only specific treatment for Fabry disease The beneficial effects of ERT on various organs/systems have been extensively evaluated The quality of life of patients receiving ERT is improved 1.2 Pompe disease – PD - Pompe disease (PD) is an autosomal recessive genetic disease that results from an alpha-glucosidase (acid maltase, GAA) deficiency caused by a mutated GAA gene, leading to glycogen accumulation excess in the lysosome This accumulation affects the normal function of other organelles in the cell and leads to cell damage It then spreads and disrupts the function of all the organs involved (eg, cardiomyopathy) The GAA gene is located on chromosome 17 at position 17q25, including 20 exons in which exon does not participate in coding To date, nearly 700 mutations in the GAA gene responsible for Pompe disease have been reported - Incidence: the incidence of the disease varies in the study populations, ranging from 1/14,000-50,000 people - Clinical manifestations in Pompe vary widely depending on the age of onset The disease can be diagnosed based on clinical symptoms, GAA enzyme activity tests, or genetic analysis tests for mutations - Similar to FD, ERT also brings high treatment efficiency for PD patients, helping to improve the quality of life and prolong life for patients Chapter OBJECT AND METHOD OF RESEARCH 3.1 Object - Criteria for patient selection: + FD: subjects with clinically suggestive symptoms (unknown cause hypertrophic cardiomyopathy detected by echocardiography or electrocardiogram), with low enzyme activity detected by newborn screening, or family members of FD’s patient + PD: subjects with clinically suggestive symptoms (muscle weakness), low enzyme activity detected by newborn screening, or members of someone in your family who has Fabry disease - Criteria for selection of family members: if GLA or GAA gene mutations are detected in patients participating in the study, pedigrees, and sampling of members of the same bloodline within generations will be made, analyze GLA or GAA gene at the mutation site found in the patient - Exclusion criteria: patients or family members did not agree to participate in the study 2.2 Study methods - Research methods: Descriptive study - Sample size: Select samples conveniently because FD and PD are rare diseases We collected: + FD: 433 patients HCM, 01 patient with low GLA activity (detected by newborn screening, 09 members of an FD’s patient + PD: 14 patients with clinically suggestive symptoms, 70 members of families which have PD patients 2.3 Time and place of study - Time: from June 2014 to June 2022 - Place: samples were taken at Hai Phong Children's Hospital, National Children's Hospital, Hanoi Heart Institute, and Green Hospital Sample analysis site: enzyme activity test was carried out at Taipei Veterans General Hospital, and gene analysis was performed at two places: Taipei Veterans General Hospital and Center of Genes - proteins research - Hanoi Medical University, all use PCR to amplify genes, directs sequencing using Sanger method, with the same primers, components and thermal cycles of the reaction 10% of the samples were analyzed at both sites to ensure the accuracy of the results 2.4 Research process and variables Collecting data on clinical and paraclinical symptoms (e.g echocardiogram, chest X-ray, ALT, CK test) using Research medical records, measuring GLA, and GAA activity, and analyzing gene sequences for patients with low enzyme activity Compared with the sequence on GeneBank to find mutations, new mutations are predicted to cause disease by bioinformatics software For subjects with genetic mutations: take samples of family members, and analyze genes at the location where mutations have been detected in the patient 2.5 Materials and chemicals Using equipment, machinery, and chemicals of reputable brands such as Qiagen (Germany), Thermo Fisher (USA), and Genzyme - Sanofi (USA) 2.6 Techniques used in the study - Measurement of enzyme activity (GLA and GAA) from dried blood spots (DBS) by tandem mass spectrometry - Extract DNA from whole blood: use Qiagen's QIAamp DNA kit (according to the manufacturer's instructions) - Carry out PCR reaction to amplify the desired gene fragment with different reaction components and heat cycles for each exon - PCR gene sequencing: Using the Sanger method on ABI 3730 XL system (Thermo Fisher) running with BigDye™ Terminator v3.1 Cycle Sequencing Kit 2.7 Data analysis - Clinical and laboratory variables were analyzed on SPSS16.0 software - Enzyme activity was calculated using specialized software according to the manufacturer's instructions - The results of gene sequencing were processed using BioEdit 7.2.6 software, and CLC Workbenches 8.0 software, then the sequences were compared on GeneBank to detect mutations Search for mutations, mutants, altered proteins, etc., using the online gene bank http://www.hgmd.cf.ac.uk/ac/index.php - Apply genealogical methods and use genealogical symbols in accordance with regulations on SPSS16.0 2.8 Ethics in research - The study was conducted with the consent of the Medical Ethics Committee of BVTEHP, the National Children's Hospital, the Scientific Council of Hai Phong Medical University, the patient, and the patient's family Chapter STUDY RESULTS 3.1 Common characteristics of the patients 3.1.1 Fabry disease - Classifying patients by age: Chart 1: Age of patients with hypertrophic cardiomyopathy Chart shows that patients with hypertrophic cardiomyopathy in this study mainly belonged to the group of 40-80 years old, the mean ± SD for the age group was 57 ± 12 years old Classifying patients by sex: In 433 patients with hypertrophic cardiomyopathy participating in the study, 417 patients were male, accounting for 97%, and only 3% of patients were female 1.1.1 Pompe - Classifying patients by age of the first onset Almost patients had early onset (less than months old), and the mean age of onset was ± 4.2 months The earliest manifestation was days old and the latest case was 17 months old - Classifying patients by sex: Of the 14 patients with clinical symptoms suggestive of Pompe disease in this study, were male and female The prevalence of the disease in both sexes is similar 1.2 Enzyme activity 3.2.1 GLA activity The GLA enzyme activity in the studied samples ranged from 0.17-9.18 μmol/h/L (1.87±0.94 μmol/h/L), mainly in the range of 0.92-2 ,97 μmol/h/l More than 60% of the patients had enzyme activity lower than the Taiwanese reference value of 2.05-7.46 μmol/h/L, of which 26 patients had the remaining enzyme activity below 10% of the value reference, accounting for 6% 3.2.2 GAA activity The results of measuring GAA enzyme activity on 14 patients with suspected Pompe in this study ranged from 0.01 to 6.8 μmol/h/L (0.91±1.26 μmol/h/L) 100% of patients participating in the study had enzyme activity decreased compared to the reference value, of which 35.7% of patients had the remaining enzyme activity less than 10% of the reference value (G Intron 1 5'UTR -12 G>A Exon SNP 5'UTR -10 C>T Exon SNP IVS4-16 A>G Intron 14 12 IVS6-22 C>T Intron 16 13 The frequency of the IVS6-22 C>T variant was the highest (48.1%), of the IVS4-16A>G variant was 44.4%, and of the 5'UTR-10 C>T variant was 37% 18.5 % of patiencarrying all variants, of which are female, are homozygous or these variants In addition, variants were detected, IVS1+17 A>G and 5'UTR -12G>A Patients with codes FB64 and VN.06 both have mutations in the coding region (exon 1) at position 178, replacing nucleotide C with nucleotide T, leading to a change in the amino acid at position 60, which is Prolin to Serin This is a new mutation that has never been published before in studies of Fabry disease Figure 3.1 Image of mutation c.178C>T, p.Pro60Ser Using the bioinformatics software SIFT, PolyPhen-2, and MutationTaster to predict the pathogenicity of this mutation all resulted in a high degree of pathogenicity When analyzing the enzyme activity results according to the gene sequencing results, we found that the group with the mutation in the coding region had the lowest enzyme activity, but when compared with the other two groups, there was a mutation in the coding region variables in the non-coding region and the group without mutations, we found that the difference was not statistically significant 10 Figure 3.7 Pedigree of patient Pom 16’s and Pom17’s family - Patient's family code Pom18 members carry mutations, including paternal grandmother (I-1) and father (II-1) of the patient carrying the mutation c.2173C>T (p.Arg725Trp), heterozygous; The patient's paternal grandfather (I-2), maternal grandfather (I-3) and mother (II-2) of the patient both carry the heterozygous c.1933G>C (p.Asp645His) mutation - Patient's family code Pom19 members carry mutations, including: grandfather (I-2) and biological father (II-3) with the same mutation c.2040+1G>T, heterozygous, maternal grandfather (I-3) ( II-4) and his brother (III-5) both carry the c.1735G>A mutation (p.Glu579Lys), heterozygous Figure 3.9 Pedigree of patient Pom18’s and Pom19’s family 11 - Patient's family code Pom20 members carry mutations, including: paternal grandmother (I-1) and biological father (II-2) with the same mutation c.1411-1414del (p.Glu471Profs), heterozygous, maternal grandmother (I-3), mother (II-2) and uncle (III-3) both carry the mutation c.1735G>A (p.Glu579Lys), heterozygous - Patient's family code Pom21 members carry mutations, including: grandfather (I-2), maternal grandfather (I-3), father's uncle and sister (II-2), aunt (II-4), biological father (II-5) , mother (II-4) and cousin (III-1) both carry the heterozygous c.625T>C mutation peoples carry heterozygous mutations, including the paternal grandmother (I-2) and father (II-2) carry c.1411-1414del (p.Glu471Profs) mutation, and the maternal grandmother (I-3), mother (II-2), and uncle (II-3) carry c.1735G>A (p.Glu579Lys), this patient received these two mutations from her parents Figure 3.10 Pedigree of patient Pom20’s family - Family of patient Pom21: 6/8 members carry mutations, including: grandfather (I-2), maternal grandfather (I-3), aunt (II-1), biological father (II-2), biological mother (II-3) and he (II-4) both carry the c.1933G>C mutation (p.Asp645His), heterozygous - Family code VN.0055 5/8 members carry the mutation, including husband and wife, fatherin-law, biological father, and daughter 12 Figure 3.12 Pedigree of patient Pom22’s and VN.0055’s family The percentage of carriers in these family The percentage of members carrying the GAA gene mutation This rate is relatively high, ranging from 70-100%, in first-degree relatives, it is over 50% in all families participating in the study, in second-degree relatives, it ranges from 50-100% 5/9 families have all members of second-degree relatives carrying the disease gene), in third-degree relatives ranging from 0-100% 3.5 Clinical and paraclinical characteristics 3.5.1 Clinical and paraclinical characteristics of FD - Patient number FB 64 carrying GLA gene mutation in the coding region is male, 56 years old The ultrasound results showed that the left ventricular mass index (LVMI) was 205 g/m2, and the left ventricular posterior wall thickness at diastolic (LVPWd) was 22.6 mm, both of which were very high This patient only exhibits features of atypical Fabry disease such as myocardial hypertrophy with symptoms of dyspnea, angina, and tachycardia, especially when working or exercising vigorously, and does not carry other features of typical Fabry disease - Patient code VN06 has low enzyme activity but no clinical symptoms The male members of the patient's maternal family all carry the gene mutation, and have low enzyme activity but have not shown clinical symptoms, and the LVMI and LVPWd indexes are within normal limits 13 3.5.2 Clinical and paraclinical characteristics of PD The clinical symptoms’ onset 10/14 patients participating in the study showed muscle weakness, 2/10 patients showed poor feeding Changes in cardiothoracic index and LVMI on chest X-ray and echocardiography In 11 patients with LVMI results, up to 10 patients have this index higher than the reference value The average LVMI of the patients was very high, in the range of 236±127g/m2 There were 6/12 patients with increased thoracic index (>60%), the mean thoracic index was 62.5±7.7% Results of measuring enzyme activity ALT, CK Both CK and ALT activities increased very high (more than times) compared to the reference value (Mean ± SD, 909.5 ± 707.8 and 287.7 ± 187.1, respectively) Chapter DISCUSSION 4.1 The common characteristics of the research subjects 4.1.1 The common characteristic of FD This study showed that patients with hypertrophic cardiomyopathy were mainly in the age group of 40 to 80 years old, especially from 50 to 59 years old (33%) This result is relatively consistent with some previous studies on screening for Fabry disease in patients with hypertrophic cardiomyopathy in the world such as the study of Nakao et al 1995, or the study of Ole Havndrup et al (2010) ), research by Albert A Hagege (2010) in France and research by David Zemánek et al (2022) Regarding the sex of the study participants: 97% of the study participants were male, out of the 26 patients with the lowest enzyme activity sequenced, the male percentage also accounted for the majority This is consistent with the rule of X-linked inheritance of Fabry disease 4.1.2 The common characteristic of PD The average age of onset of patients in the study was 5±4.2 months, most of which were under year of age, this result is consistent with previous 14 publications on IOPD where the age of onset is usually about months , the age of diagnosis is usually months Gender characteristics: male/female ratio in our study is 1/1 This is completely consistent because Pompe is an autosomal recessive genetic disease, so there is no sex difference 4.2 The change in enzyme activity 4.2.1 GLA activity of patients with hypertrophic myocardial The GLA activity of the patients in our study was much lower than the Taiwan reference range, possibly because the study participants were patients with unexplained hypertrophic myocardial, so there was no representative Our results are also lower than those of Nakao et al (1995) or the results of B Sachdev et al (2002) on the same subjects with hypertrophic myocardial, possibly due to the use of the different methods of determining GLA enzyme activity (parallel mass spectrometry and standard fluorescence spectrometry with 4-methyl-umbelliferyl a-D-galactopyranoside as substrate) In this study, there were 02 subjects of the same age, carrying the same mutation but very different enzyme activity, this result is similar to the result of D Zemáneket (2022) on male patients with the same have the same mutation and the same age but have different enzyme activities 4.2.2 GAA activity of patients with clinical symptoms suspected of having Pompe disease All pediatric patients with clinical symptoms suggestive of Pompe disease had GAA activity less than the reference value, of which patients had enzyme activity 3% lower than the reference value These patients all showed their first symptoms before 6-month-age This is completely consistent with the description in previous studies; with IOPD, GAA activity A have been recognized as GLA gene 16 SNPs However, there was no statistically significant difference in enzyme activity in patients with these variants compared with patients who did not carry the mutation or carried the mutation in the coding region It is possible that these mutations occur in the intron region or the 5'UTR region, which is not involved in the transcription of proteins, specifically the GLA enzyme However, science is evolving and the role of many DNA sequences is still a mystery to human understanding, so whether these mutations have any effect on the phenotype of individuals or not further research 4.3.2 GAA gene mutations The GAA enzyme is composed of five domains, each of which has different roles in the transport, maturation, and catalytic activities of the GAA enzyme, so mutations in different domains will have different consequences A study by Kanako Sugawara et al in 2009 showed that amino acid substitutions that cause abnormalities in the maturation or transport of the GAA enzyme that cause Pompe disease can be found on all five domains of this enzyme They are distributed from the core to the surface of the enzyme molecule, and the predicted structural changes vary from large to very small In this study, we detected 10 mutations (including missense mutations, frameshift mutations, and splicing mutation) and SNP Missence mutation c.1933G>C (p.Asp645His) This mutation is the one with the highest frequency in our study (7/14 cases, accounting for 50%) The c.1933 mutation replaces G with C, changing the amino acid Aspartate into Histidine at position p.645 of exon 14 This mutation takes place in the highly conserved region of the GAA gene The substitution of Aspartate into Histidine at the p.645 position, which is the catalytic region of the GAA enzyme, has been shown to significantly reduce GAA activity, homozygous patients with this mutation exhibit a severe phenotype This mutation has been reported in previous studies in both IOPD and LOPD patients, homozygous or double heterozygous, in association with various heterozygous mutations such as the c.1933G mutation >C or mutant c.1411-1414del in Lukana's study (2019) In this study, the homozygous c.1933 G>C (p.Asp645His) mutant was found in 03 different IOPD patients, all with very low enzyme activity The 17 combination of this heterozygous mutation with the other heterozygous mutation such as c.1933G>A mutation (p.Asp645Asn), the p.Leu246fs*22 mutation, or the c.2173C>T mutation (p.Arg725Trp) has been reported as pathogenic mutations in many previous studies in both IOPD and LOPD patients Patients carrying these compound mutations all have low enzyme activity and early onset (less than year of age) Missence mutation c.625T>C (p.Tyr209His) This mutation has the second highest occurrence frequency in our study with 4/14 cases The mutation at position c.625 replaces T with C, changing the amino acid Tyrosine to Histidine at position p.209 on exon This is a mutation that has never been reported in the database of GAA gene mutations The c.625T>C (p.Tyr209His) mutation takes place at the N-terminal β-sheet domain, which is responsible for the processing of signal peptides, so this mutation may affect the transport of precursors into the lysosomes, thereby affecting enzyme synthesis The tools PolyPhen-2 and MutationTaster all predict this mutation can cause disease to different degrees, but SIFT predicts this is benign This mutation is seen in patients with codes Pom8.0, Pom10.0, and Pom21.0 all in compound heterozygous mutations, especially, patients whose codes Pom8.0 and Pom10.0 have identical genotypes, but these patients have very different GAA enzyme activity and very different age of onset, therefore experimental studies are needed to prove the pathogenic mechanism of this mutation Others missence mutation The c.1745G>A (p.Glu579Lys) mutation was reported in a 1993 study by Herman in case of LOPD as one of two mutations that cause decreased intracellular transport and maturation of the GAA enzyme that does not affect the synthesis of precursors of this enzyme The heterozygous c.2563 G>C (p.Gly855Arg) mutation was described in a patient with Pompe disease along with the c.111A>T mutation in the study by Xi Chen et al on 25 pediatric patients with Pompe disease may have an early onset in Taiwan, however, this mutation is classified as pathogenic because the mechanism related to the decrease in enzyme activity or the clinical symptoms of the patient is not clear