| Duchenne muscular dystrophy/Becker muscular dystrophy (DMD/BMD) is one of the most common X-linked recessive lethal genetic diseases. DMD acquired its name because it was first reported by Duchenne et al.(1868). The incidence of this disease in the general population has reached1/3,500live male births, and DMD is a commonly occurring primary muscle disease with a poor prognosis. Typical clinical features of this disease include progressive muscular atrophy and muscle weakness associated with pseudohypertrophy of the gastrocnemius muscle of the lower leg. DMD typically affects adolescent males, who generally lose the ability to stand and walk before reaching12years of age; DMD patients often die by20years of age due to heart failure or respiratory failure caused by cardiac and respiratory muscle weakness. The disease greatly impacts the healthy growth of adolescent males and has brought heavy mental and economic burdens to patients’ families and to society as a whole.To date, no effective treatments for this disease exist. Therefore, the verification of probands, the prenatal diagnosis of DMD carriers, and the elimination of the birth of affected children remain key aspects of eugenics-based genetic approaches for the prevention of DMD in China and around the world.Before the application of genetic diagnostic techniques, the clinical diagnosis of DMD relied on typical symptoms and signs in combination with other secondary screening techniques, such as electromyography (EMG), assessments of enzyme biochemistry, and muscle biopsy. Among these screening approaches, EMG results that indicate myogenic damage and assessments of enzyme biochemistry that reveal significant increases in muscle enzyme activity represent relatively reliable bases for DMD diagnoses. However, these diagnostic approaches do not exhibit high specificity but, instead, generate false positive and false negative results. Therefore, the clinical applications of these two types of screening approaches are somewhat limited.The DMD gene is the largest known human gene, with79exons and78introns. This gene exhibits a high mutation frequency and diverse mutant forms. Mutations in the DMD gene are the molecular genetic basis of DMD/BMD pathogenesis. Deletion mutations, duplication mutations, and point mutations are the main types of mutations of the DMD gene.Primary methods of genetically diagnosing DMD included Southern blot hybridization, multiplex polymerase chain reaction (PCR) techniques, short tandem repeat (STR)-PCR, and reverse transcription PCR, among other approaches. Chamberlain et al. designed nine pairs of multiplex PCR primers that could detect deletions in the DMD gene in80%of DMD patients. Beggs et al. added nine additional pairs of multiplex PCR primers to the sets of primers that Chamberlain et al. had designed for DMD detection, and in combination, these18pairs of primers could detect deletions in the DMD gene in98%of DMD patients. Prior et al. first applied quantitative PCR approaches for the diagnosis of heterozygous DMD carriers; in addition, STR-PCR technologies can be used for linkage analyses of non-deletion pedigrees.In the past, we have used various methods for the prenatal diagnosis of DMD, including gender diagnoses, multiplex PCR-based analyses of deletion mutations, and linkage analyses of STR haplotypes. These methods exhibit certain limitations and are not well adapted to the requirements for prenatal diagnoses. Gender diagnosis alone could lead to the elimination of normal male fetuses. Notably, the intragenic exchange rate for the DMD gene may be as high as11%. Thus, linkage analyses of STR haplotypes could theoretically exhibit a false-positive or false-negative rate of up to11%. Therefore, the results from these types of haplotype linkage analyses can only be regarded as probabilistic diagnoses. In addition, at times, linkage analysis cannot be performed due to lack of information regarding probands and heterozygotes or because available pedigrees are overly small. Multiplex PCR-based approaches for DMD diagnoses do not produce sufficient quantities of genetic information to span the79exons of the DMD gene; thus, these approaches can easily lead to misdiagnoses. Moreover, these approaches cannot detect duplication mutations, heterozygous carriers, or point mutations.The objectives of our study are not only to establish a clinically feasible genetic approach for the diagnosis and prenatal diagnosis of DMD that is more efficient and less time-consuming than current diagnostic approaches for this disease but also to explore the methods and timing of early-stage prenatal diagnoses of DMD.DMD is an X-linked recessive genetic disease; thus, in theory, DMD should predominantly affect males. However, a handful of female carriers of DMD evince clinical symptoms of this disease due to the occurrence of various unusual phenomena, such as secondary mutations. These female DMD patients are often misdiagnosed with autosomal recessive limb-girdle muscular dystrophy, which exhibits symptoms that are similar to DMD symptoms. In recent years, studies have indicated that the pathogenesis of DMD among female patients may involve X-chromosome inactivation and other types of epigenetic regulation. Therefore, the study of the molecular mechanisms underlying DMD in female patients possesses important clinical value and scientific significance.To date, no specific treatments exist for DMD; thus, attempts to discover effective therapeutic approaches for DMD have long been a hot topic of academic research in the field of neuropathy. Induced pluripotent stem cells (iPSCs), which exhibit good immunocompatibility, are regarded as a potential source for the development of treatments of various diseases; moreover, these cells also provide an ideal model for the study of disease mechanisms. Thus, iPSCs appear to be a promising avenue to explore for discovering treatments for diseases of the nervous system. In2008, the Harvard University researchers Dimos et al. published an article in Science that detailed the ways in which they not only obtained iPSCs from the somatic cells of an82-year-old amyotrophic lateral sclerosis (ALS) patient with point mutations in the superoxide dismutase1, soluble (SOD1) gene by viral transduction, viral induction, and reprogramming but also induced the iPSCs to differentiate into motor neurons that had been damaged in the patient due to ALS. This discovery has markedly altered the difficult scenario of treating ALS patients. This finding also implies that it is possible to prepare and repair iPSCs for a patient by reprogramming the patient’s somatic cells; these iPSCs can then be utilized for autologous transplantation treatments that could achieve good therapeutic effects with markedly lower levels of immune rejection than current transplantation approaches could provide. Therefore, in this study, a preliminary investigation of the establishment of iPSC lines was conducted. Chapter1The establishment of an appropriate technology platform for the molecular diagnosis of deletion and duplication mutations of the DMD gene and the application of this platform for perinatal diagnosisObjectivesTo establish an appropriate experimental method for comprehensively screening all79exons of the DMD gene, focusing on deletion and duplication mutations; this method would be used for general and prenatal diagnoses of DMD patients and heterozygous carriers, replacing the current diagnostic approaches that only utilize probabilistic and gender-based diagnoses.To explore the possibility of genetically detecting DMD by chorionic villus sampling, which would permit prenatal diagnoses of DMD to occur at earlier stages than current techniques would allow.To perform whole genome amplification for a single embryonic cell and explore experimental methods for the diagnosis of DMD prior to embryo implantation.MethodsData sources:1. The examined cases of this study were mainly derived from our hospital. These cases consisted of probands with typical clinical manifestations of DMD for whom examinations of serum muscle enzymes, EMG, muscle biopsy, or other assessment approaches had supported a DMD diagnosis and excluded other neuromuscular genetic diseases.2. Amniotic fluid or chorionic villus samples were obtained from DMD carriers during pregnancy.3. After obtaining informed consent, single embryonic cells were acquired from surplus embryonic blastomeres.Experimental methods:1. Proband samples were analyzed using the multiplex ligation-dependent probe amplification (MLPA) method with the P034and P035SALSA probe mixes (MRC Holland). The samples were subjected to denaturation, hybridization, and ligation reactions followed by PCR amplification and the capillary electrophoresis analysis of the resulting products, allowing for the detection of deletion and duplication mutations in the79DMD exons. This approach was compared with the multiplex quantitative PCR method.2. A total of155pregnant women who had been verified to be heterozygous carriers of deletions or duplications in the DMD gene were examined, and the MLPA method was applied to produce prenatal diagnoses of at-risk fetuses using amniotic fluid or chorionic villus samples.3. During the chorionic villus sampling process, maternal blood from the carriers was extracted. DNA-STR genotyping was performed for chorionic villi samples and maternal samples using the GoldeneyeTM DNA identification system to distinguish among individuals.4. The REPLI-g Midi Kit was used to perform whole-genome amplification-multiple displacement amplification (WGA-MDA) of single embryonic cells. The WGA-MDA products were validated by the amplification of DMD-STR loci.Results1. Prenatal diagnoses of the fetuses of155pregnant women who carried deletions or duplications in the DMD gene detected27fetuses that were diagnosed with DMD,28fetuses that were diagnosed as carriers of mutations in the DMD gene, and100normal fetuses. The155examined fetuses included72male fetuses. The27 fetuses that had been diagnosed with DMD constituted38%of the examined male fetuses (27/72), and the45normal male fetuses that were assessed in this study constituted63%of the total number of examined male fetuses (45/72). A total of83cases of female fetuses were examined. The28fetuses that had been diagnosed as carriers of mutations in the DMD gene constituted34%of the examined female fetuses (28/83), and the55normal female fetuses that were assessed in this study accounted for66%of the total number of examined female fetuses (55/83). The pregnancies of women with fetuses that had been diagnosed with DMD were terminated, whereas the pregnancies of women with normal fetuses or fetuses that were diagnosed as carriers of DMD gene mutations were continued. Subsequent follow-up examinations were consistent with the prenatal diagnoses r%arding the DMD gene.2. The MLPA approach could generate information for all79exons of the DMD gene, whereas the44multiplex quantitative PCR test results obtained through two experiments (with nine primer pairs in each experiment) only produced information for18exons of this gene.3. WGA-MDA was performed for eight single embryonic cells, and the results of this analysis were verified by the amplification of DMD-STR loci. The WGA-MDA amplification yielded good results, with a quantity of amplified product that met the requirements for subsequent detection of the target gene and pre-implantation genetic screening.Conclusions1. The application of MLPA provides the most effective known genetic approach for the general diagnosis and prenatal diagnosis of deletion and duplication mutations in the DMD gene. In particular, this diagnostic technique is not only capable of identifying whether a DMD gene has deletion mutations or duplication mutations but can also distinguish between DMD patients and DMD carriers.2. The MLPA method may be combined with DNA-STR genotyping technology to analyze chorionic villi samples for the early diagnosis of DMD. This combined approach can identify the presence of maternal tissue pollution in samples and thereby avoid misdiagnoses.3. DMD gene mutations are mainly distributed between exon45and52, there are a mutation hot spots.4. The mutation rate of DMD exons duplication have significant difference from exon deletion, which is inherited from the mother more, fewer new mutations.5. Individual embryonic cells can be analyzed via WGA-MDA, which increases the quantity of template that is available, thereby overcoming the shortcoming that a single cell would otherwise only provide sufficient material for one detection experiment and providing experimental preparations that may be utilized to perform preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS) for DMD.Chapter2The establishment and application of molecular diagnostic techniques to detect DMD point mutationsObjectives:Results from the first chapter of this investigation indicate that MLPA technology can detect the deletions and duplications in the DMD gene that exist in70%of DMD patients. However, in the remaining DMD patients, who constitute approximately30%of the total number of DMD patients, DMD is caused by point mutations in the DMD gene that cannot be detected by MLPA approaches. At present, because of the large size of the DMD gene, there are no medical institutions in China that can routinely detect point mutations in the DMD gene; therefore, there is no opportunity to obtain pedigrees of point mutations for use in prenatal diagnoses.This study attempts to develop efficient sequencing methods that can detect point mutations in the79exons of the large DMD gene and thereby provide an approach that can diagnose DMD patients with point mutations who are currently unable to be genetically diagnosed with DMD through conventional clinical methods. It is hoped that these methods will ultimately increase the proportion of DMD cases that are genetically diagnosable as DMD from the current ratio of70%to90%or more. We also explore the use of the high-resolution melting (HRM) method for the preliminary screening of point mutations in the DMD gene. Furthermore, to identify single-exon deletions and point mutations, this study combines the PCR and the MLPA methods with sequencing; this combined approach can avoid misdiagnoses and provide a theoretical basis for gene repair therapies.MethodsData source:The examined cases of this study were mainly derived from our hospital. Probands exhibited typical clinical manifestations of DMD, and examinations of serum muscle enzymes, EMG, or muscle biopsies were utilized to support the DMD diagnosis and exclude other neuromuscular genetic diseases. The MLPA method confirmed that there were no deletion or duplication mutations in the DMD gene exons of the examined patients.Experimental methods:1. The Qiagen method was used to extract genomic DNA from peripheral blood samples from the examined DMD patients.2. The MLPA approach was used to exclude patients with any deletion or duplication mutations in their DMD gene exons.3. All79exons were sequenced in the following manner. PCR amplification was performed on the genomic DNA samples from the study subjects. Subsequently, the PCR products were purified, sequencing reactions were performed, and the sequencing PCR products were purified. An ABI3100Genetic Analyzer was then used to assess these purified sequencing products through capillary electrophoresis, and the results from this process were analyzed and processed with the GeneMapper ID v3.1and Mutation Surveyor software packages to obtain sequencing results.4. A LightScanner HRM system was used to detect known point mutations and certain unknown point mutations in the examined samples.5. MLPA results indicating single-exon deletions in patients were subjected to verification through PCR and DNA sequencing.Results1. We performed genetic sequencing of blood samples from16probands and found that in13of these patients, a single nucleotide substitution had occurred in the DMD gene. Among the remaining three probands who were examined, one proband had a DMD gene that was missing a single nucleotide, one proband had a DMD gene that was missing two nucleotides, and the final proband had a DMD gene with an insertion of5nucleotides. These point mutations resulted in premature termination codon mutations, frameshift mutations, and other deleterious alterations of the DMD gene.2. In total,37cases that the MLPA method had diagnosed as single-exon deletions from the DMD gene were examined, and in30of these cases,81.08%of the examined cases, an exon deletion in the DMD gene was detected through PCR and sequencing approaches. In the remaining seven cases, which accounted for18.92%of the examined instances of alleged single-exon deletion, the PCR method did not identify deletions, and sequencing provided conclusive evidence that exon point mutations were present in these seven cases. 3. HRM analyses that were performed for DNA samples from ten cases revealed peaks to indicate point mutations for exons of100-300bp in size; however, in contrast to expectations, these peaks failed to appear for exons of other sizes.Conclusions1. The genomic sequencing method is a reliable detection method for point mutations in the DMD gene. After the MLPA method has been utilized to exclude DMD patients with deletion or duplication mutations in this gene, the sequencing of the complete set of DMD exons could be utilized to genetically diagnose DMD in the remaining patients, who have DMD point mutations. It can be applied to prenatal diagnosis.2. A determination of a single-exon deletion by the MLPA approach must be combined with the application of PCR and sequencing methods, which can identify whether the MLPA results reflect a true deletion or a point mutation and thereby improve upon the accuracy of the MLPA approach for the detection of single-exon deletions in the DMD gene.3. High-resolution melting curves can be used to screen for point mutations. However, under the conditions that were employed in this study, this method is only effective to detect point mutations in exons of100-300bp in size. Thus, further exploration is required to improve methods for screening for point mutations.Chapter3The study of DMD pathogenesis in females and the establishment of iPSC lines from amniotic fluidObjectivesDMD/BMD is an X-linked recessive genetic disease; thus, in theory, this disease should predominantly affect males. Clinical symptoms of this disease in females appear in a handful of carriers due to various phenomena, such as secondary mutations. Therefore, female patients with this disease are often misdiagnosed with autosomal recessive limb-girdle muscular dystrophy, which exhibits symptoms that are similar to the manifestations of DMD/BMD. In recent years, studies have shown that the pathogenesis of female patients might involve epigenetic mechanisms, such as X-chromosome inactivation. Therefore, the study of the molecular mechanisms of DMD pathogenesis in female patients has important clinical value and scientific significance. In this study, to detect the presence of small deletions/duplications in the genomes of female DMD patients, high-resolution Affymetrix CytoScan HD arrays are utilized to perform genome-wide analyses of these patients. Methylation-specific PCR is employed to detect X-chromosome inactivation and examine the correlation of this inactivation to the pathogenesis of DMD in female patients.There are currently no specific treatments for DMD; instead, only symptomatic treatments and supportive care exist for this disease. The search for effective treatments for DMD has long been a hot topic of academic research in the field of neuropathy. iPSCs, which exhibit good immunocompatibility, are regarded as a potential source for the development of treatments of various diseases; moreover, these cells also provide an ideal model for the study of disease mechanisms. Many cell components exist in amniotic fluid; in fact, at present, the amniotic fluid is thought to include a heterogeneous population of cells that have been derived from fetuses and amniotic membranes. Amniotic fluid cells, which consist of a variety of cells from different sources, exhibit excellent pluripotency potential and can be induced to differentiate into cells of all embryonic germ layers. This study seeks to use the two factors of octamer-binding transcription factor4(Oct4) and Kruppel-like factor4(Klf4) to induce the re-programming of amniotic fluid cells and thereby obtain iPSCs; this process would provide a suitable research foundation for developing stem cell therapies for DMD and establishing models of this disease.MethodsData sources:1. Data were obtained from the four female DMD patients who have been treated in our hospital since2006.2. Amniotic fluid cell samples were collected from a woman who had undergone a medically recommended amniocentesis when she was20weeks pregnant with twins. After prenatal diagnostic procedures using the sampled amniotic fluid from this woman had been completed, the remaining amniotic fluid was employed for this study after obtaining informed consent from the donor couple and approval from the ethics committee of the Third Affiliated Hospital of Guangzhou Medical College.Experimental methods:1. The Qiagen method was used to extract genomic DNA from the peripheral blood samples of female DMD patients. Analytical procedures and quality control were performed in accordance with the operating protocols for the Affymetrix CytoScan HD array.2. To detect the status of X-chromosome inactivation in the examined patients, genomic DNA samples from these patients were treated with sulfates and then amplified with methylation-specific primers. The amplification products were analyzed via capillary electrophoresis.3. The iPSCs were prepared as follows. First, the examined amniotic fluid cells were collected, passaged, and infected. A primary culture of human amniotic fluid-derived cells (hAFDC)-iPSCs was created and passaged. These cells were then subjected to reverse transcription (RT)-PCR detection of the expression of pluripotency genes, the detection of alkaline phosphatase (AP), immunofluorescence staining, the detection of in vitro differentiation capacity, the detection of in vivo differentiation potential, and karyotype analysis.Results1. Genome-wide copy number analyses were performed for one female DMD patient and her parents. These analyses revealed a DMD gene in this patient with the duplication of exons2-37(i.e., an abnormally high copy number); this result was consistent with findings from the MLPA approach. The patient’s mother possessed a DMD gene with a duplicated10kbp fragment in intron4. Single nucleotide polymorphism (SNP) genotyping suggested that the patient had obtained her allele containing the aforementioned-duplication mutation in DMD from the mother.2. Genome-wide copy number analysis was performed to assess one female DMD patient for whom MLPA approaches had failed to find abnormalities. This analysis revealed an abnormally high copy number for the examined patient for a31kbp fragment in the Xp21.1region that overlapped with part of exon47of the DMD gene. In addition, this patient exhibited an abnormally high copy number for a2kbp fragment in intron9of the DMD gene.3. Retroviruses containing the hOct4and hKlf4genes were used to infect the amniotic fluid cells. At six days after this infection, undifferentiated hAFDC-iPSC clones exhibited strong positive signals in AP detection analyses. Cells with clone-like growth were picked and cultured in feeder-free medium. The undifferentiated cells formed densely packed cell masses with clear borders. The karyotypes of the cells were examined every ten passages to assess the hAFDC-iPSCs. These cells have undergone40passages. During the passaging process, the two cell lines were both able to maintain a normal karyotype of46, XX. hAFDC-iPSCs and the human embryonic stem cell line FY-hES-1exhibited rather similar expression levels of pluripotency genes; in particular, the Oct4and Nanog genes were highly expressed in both of these cell types, whereas neither of these genes was expressed in differentiated amniotic fluid cells. The hAFDC-iPSCs clones were strongly positive for the transformer (TRA)-1-60surface antigen and positive for NANOG/OCT4expression, indicating that these clones existed in an undifferentiated state.Conclusions1. Double mutations is one of the reasons for the female DMD patient.2. DMD gene mutation combined chromosomal abnormalities is another reasons.3. High-density gene arrays were utilized to investigate the pathogenesis of female DMD patients. These analyses determined that one female DMD patient had an abnormal copy number for portions of the DMD gene and that this phenomenon might involve exons of this gene. A female DMD patient with a duplication of exons2-37of the DMD gene appears to have inherited this abnormal allele from her mother. One of her mother’s DMD genes contains small intronic fragment repeats, suggesting that this DMD gene might be unstable. Certain female DMD patients exhibit secondary mutations of their DMD genes.2. The two factors of OCT4and KLF4were utilized to induce the reprogramming of amniotic fluid cells to obtain iPSCs, thus establishing a suitable foundation for the development of DMD stem cell therapies and for the establishment of models of DMD. |
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