定量多重PCR在筛查DMD携带者和产前诊断中的应用

基于抗肌萎缩蛋白基因(DMD)多个外显子进行对数增长期多重PCR(多聚酶链反应)产物定量分析的原理,本研究在改进Ioannou等方法基础上建立了假性肥大型肌营养不良症(DMD/BMD)定量多重PCR(QM-PCR)技术,应用于携带者筛查和产前诊断.运用多重PCR技术对12个DMD/BMD家系先证者的检测表明缺失一个或一个以上外显子的家系有7个(58.3%).QM-PCR证实7名缺失型患者中6名母亲是与缺失型患者相同的携带者(86.7%).1例检测出外显子缺失的DMD患者,其母亲及外祖母的外周血淋巴细胞DNA未发现任何DMD基因缺失,可以排除为携带者(13.3%).对一名肯定携带者妊娠9周的胎儿进行了性别测定和DMD检测,确诊胎儿为男性DMD患者.一例可能携带者在排除携带者基础上,进一步对妊娠胎儿产前诊断确诊为正常女婴.本研究的结果进一步证实了该技术筛查DMD携带者的可靠性.将本技术与其它技术相结合可明显改进DMD家系中携带者诊断的准确性,并可应用于谱系及多态性信息不详家系的携带者诊断.该技术对防止缺失型DMD患儿和携带者的出生有重要的意义.     

Rapid method for targeted prenatal diagnosis of Duchenne muscular dystrophy in Vietnam

ObjectiveSince there is no effective curative treatment for Duchenne muscular dystrophy (DMD), prevention mostly depends on genetic counseling and prenatal diagnosis. About two-thirds of the affected patients have large deletions or duplications, which can be detected by multiplex ligation-dependent amplification (MLPA). The remaining cases include small mutations, which cannot be easily identified by routine techniques. In such cases, linkage analysis may be a useful tool for prenatal diagnosis. Here we compared results obtained from linkage using short tandem repeats (STRs) with those by MLPA and sequencing analysis.Materials and methodsEight Vietnamese pregnant women at risk of having a baby with DMD and requesting prenatal diagnosis were recruited in this study. MLPA and direct sequencing were applied to screen large rearrangements and point mutations in the dystrophin gene in the DMD probands and the fetal samples. STR linkage was also performed to analyze fetal mutation status.ResultsBy MLPA and sequencing analysis, five DMD patients showed deletions of the dystrophin gene, and no deletions of exons were detected in seven amniotic fluid cell samples; one patient harbored the out-of-frame small deletion of exon 43, which was also found in the fetal sample of this family. STR analysis revealed the transmission of a mutant allele inside each family.ConclusionOur results suggest that the combination of STR and MLPA could be a rapid, reliable, and affordable detection protocol for determination of the carrier's status and prenatal diagnosis of DMD in a developing country such as Vietnam.     

Detection of a novel dystrophin gene mutation through carrier analysis performed during prenatal diagnosis in a case with intragenic recombination

OBJECTIVES: To report a multi-technical approach to Duchenne muscular dystrophy (DMD) mutation testing through carrier analysis, in the prenatal diagnosis of a male foetus without a known mutation segregating in the family and with inconclusive results of linkage analysis. METHODS: Haplotype analysis with the DMD region markers for assigning the carrier status of the mother and for prenatal diagnosis of foetal DNA; semiquantitative multiplex analysis of maternal and foetal DNA for the promoter and for 34 exons of the DMD gene; sequencing analysis of the maternal and foetal DNA for confirmation of the results. RESULTS: Because of an intragenic recombination of the DMD gene in foetal DNA, haplotype analysis gave inconclusive results. Semiquantitative PCR analysis displayed a pattern compatible with a heterozygous exon 60 mutation in the mother's DNA, while foetal DNA showed a normal migration pattern. Sequencing analysis confirmed the presence of a novel 7 base-pair deletion in exon 60 of the DMD gene in the mother and excluded the deletion in the foetus. CONCLUSION: Semiquantitative PCR results allowed the DMD mutation detection in the mother and the exclusion in the foetus, showing its crucial importance in prenatal diagnosis in those cases where linkage analysis is not conclusive.     

Multiplex ligation-dependent probe amplification identification of deletions and duplications of the Duchenne muscular dystrophy gene in Taiwanese subjects.

BACKGROUND/PURPOSE: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive disorders caused by mutations in the DMD gene. We intended to determine the distribution of DMD gene deletions and duplications in local Taiwanese male patients and potential female carriers. METHODS: A total of 102 unrelated subjects, including 89 unrelated DMD/BMD male patients and another 13 unrelated potential female carriers, were recruited for this study. Multiplex ligation-dependent probe amplification (MLPA) was employed to detect DMD gene deletions and duplications in the 102 subjects. RESULTS: MLPA was informative in 60.7% (54/89) of these patients, identifying deletions in 36.0% (32/89) and duplications in 24.7% (22/89) of these patients. This assay revealed deletions in 30.8% (4/13) and duplications in 30.8% (4/13) of the 13 potential carriers. Deletions and duplications were detected in 35.3% (36/102) and 25.5% (26/102) of a total of 102 affected families, respectively in this series. The "hotspot" regions of the duplications were close to those of the deletions. CONCLUSION: MLPA was proven to be a powerful tool for the detection of DMD gene deletions and duplications in male patients and female carriers. There was a relatively lower frequency of deletion and a higher frequency of duplication of DMD gene in this population compared to previous reports.     

杜氏肌营养不良症产前基因诊断的初步探讨

目的 对杜氏肌营养不良症（duchenne muscular dystrophy，DMD）家族史的胎儿进行dystro-phin基因缺失型的产前诊断，并探讨其产前诊断流程。方法 对3例有DMD家族史的胎儿。利用羊水细胞培养行染色体核型分析及B超检查确定胎儿性别；利用脐带血穿刺标本，应用多重聚合酶链式反应（multiple polymerase chain reaction。mPCR）技术。结合生化检测指标，进行DMD基因缺失型的产前诊断。结果 3例高危胎儿确诊为男性胎儿。存在基因缺失。相应肌酶有不同程度的升高，诊断为DMD患儿。结论 在结合多种临床实验室检查的基础上，mPCR是可应用于DMD的产前诊断。该技术也存在局限性。如只能检测男性胎儿基因缺失型突变。     

Dystrophin gene deletion in Chinese Duchenne/Becker muscular dystrophy patients via multiplex DNA amplification.

Duchenne/Becker muscular dystrophy (DMD/BMD) is a progressive muscle-wasting disease. The dystrophin gene responsible for the disease is the largest human gene ever cloned and is prone to gross gene deletion in two "hot spot" regions. Using nine pairs of oligonucleotide primers deduced from the two regions, we have screened 23 unrelated Chinese DMD/BMD patients by multiplex polymerase chain reaction. Nine (39%) patients were noted to have gene deletion, one in the 5' terminus and eight in the distal half of the gene. The incidence is similar to that reported in other large series mainly on Caucasian patients. The "hot-spot" regions also seem to be present in Chinese patients. Multiplex gene amplification for deletion analysis is useful in the diagnosis of patients with neuromuscular diseases and is an important aid in the prenatal diagnosis and genetic counseling of at-risk families.     

Prenatal diagnosis of de novo DMD duplication by multiplex ligation-dependent probe amplification (MLPA) after noninvasive prenatal screening (NIPS) at 11 gestational weeks

ObjectiveDystrophinopathy is an X-linked recessive muscular dystrophy caused by mutations in the DMD gene. Herein we describe the prenatal detection of DMD gene mutations in a patient with no family history, by multiplex ligation-dependent probe amplification (MLPA) after noninvasive prenatal screening (NIPS).Case reportA 41-year-old woman underwent NIPS owing to an advanced maternal age. A copy number variation was detected in the maternal X chromosome, and uninformative results were obtained for the fetal sex chromosomes. Following amniocentesis, a duplication was identified in exons 1–29 of the dystrophin gene by MLPA. After interviewing her family members it was confirmed that the patient is a de novo carrier of DMD duplications, and her daughter is a carrier of the same mutation.Conclusionhis is the first case report to describe the prenatal diagnosis of duplications in the DMD gene by MLPA following NIPS in a patient with no family history.     

不同表型肌营养不良症患者抗肌营养不良蛋白基因缺失类型的研究

目的探讨我国东北地区杜氏型肌营养不良症（DMD）及贝克型肌营养不良症（BMD）患者抗肌营养不良蛋白基因缺失类型分布与表型的关系,并用于产前基因诊断。方法采用多重PCR法检测124例来自东北地区的DMD（106例）及BMD（18例）男性患者的抗肌营养不良蛋白基因缺失情况,并对30例高危胎儿行产前抗肌营养不良蛋白基因缺失检测。结果124例患者中,抗肌营养不良蛋白基因缺失检出率为49％（61／124）,其中41例（41／61,67％）缺失分布于外显子45—53,13例（13／61,21％）缺失分布于外显子8—19,5例（5／61,8％）在上述两个外显子缺失区内均有缺失,2例（2／61,3％）缺失分布于外显子34和43;缺失型患者中有9例发生整码缺失（为BMD患者）,49例发生移码突变（为DMD患者）。30例高危胎儿中,17例为男性胎儿,其中10例为抗肌营养不良蛋白基因缺失型,缺失位点与先证者相同;13例为女性胎儿,无一例抗肌营养不良蛋白基因缺失。结论DMD及BMD患者抗肌营养不良蛋白基因缺失主要分布于两个区域,外显子8附近区域可能是东北地区该基因缺失高发区;缺失类型与临床表型有一定的关系,当基因发生整码缺失时,临床表型为BMD,而发生移码突变时,临床表型为DMD。     

Pitfalls in prenatal diagnosis of DMD due to placental mosaicism of the X-chromosomes: prenatal and postnatal findings in a fetus with a deletion of exons 67-71 of the dystrophin gene

Prenatal diagnosis of Duchenne and Becker muscular dystrophy (DMD) is performed as a routine procedure in many laboratories. The major potential problem is an incorrect diagnosis that could be obtained due to contamination with maternal tissue. We report a case of mosaicism of the X-chromosomes confined to the placenta as a possible source of confusing results in prenatal diagnosis of DMD. To the best of our knowledge, this is the first reported case of this problem in a prenatal DMD diagnosis.     

Small mutations of the DMD gene in Taiwanese families

BACKGROUND/PURPOSE: Duchenne/Becker muscular dystrophies are X-linked recessive disorders caused by mutations in the Duchenne muscular dystrophy (DMD) gene. We aimed to demonstrate the small mutation patterns of the DMD gene in Taiwanese subjects. METHODS: We sequenced all 79 exons of the DMD gene in 33 unrelated Taiwanese families in which large-scale deletions and duplications had been excluded by multiplex ligation-dependent probe amplification. RESULTS: Direct sequencing detected 23 different mutations from 26 families, including 15 novel mutations and eight previously reported ones. The 15 novel mutations consisted of seven substitutions (c.1238C>G [p.S413X], c.2971G>T [p.E991X], c.3172C>T [p.Q1058X], c.7402G>T [p.E2468X], c.8022C>G [p.S2605X], c.10018T>C [p.C3340R], c.10546G>T [p.E3516X]), six small deletions (c.2202delG [p.A668fsX676], c.2268delC [p.F756fsX759], c.4611delT [p.N1537fsX1545], c.4856_4857delAA [p.K1619fsX1621], c.6638delT [p.L2213fsX2220], c.9457delT [p.C3153fsX3154]), and two small insertions (c.4351insA [p.L1451fsX1468], c.10493_10495insAAT [p.L3498X]). Twenty-two of the 23 pathologic changes disrupted the translational reading frame (13 nonsense, 7 frameshift, 2 splice-site change), whereas only one was a missense variant with known pathogenic nature. Two previously reported mutations, c.8038C>T [p.R2680X] and c.10108C>T [p.R3370X] were detected in two and three unrelated families, respectively. CONCLUSION: Most identified mutations either led to a predictable premature stop codon or resulted in splicing defects, which caused defective function of dystrophin. Our findings extend the mutation spectrum of the DMD gene. Molecular characterization of the affected families is important for genetic counseling and prenatal diagnosis.     

Rapid and powerful decaplex and dodecaplex PGD protocols for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a common childhood lethal X-linked recessive disorder, resulting from deletions, duplications and point mutations in the dystrophin gene. Single-cell protocols for preimplantation genetic diagnosis (PGD) still remain challenging due to the enormous size of the gene and the high risk of intragenic recombination, limitations that often lead to sex determination and selection of female embryos. This study describes direct and rapid decaplex and dodecaplex polymerase chain reaction protocols enabling the analysis of five or seven exons and four microsatellite markers scattered along the dystrophin gene, chosen to be located in the two deletion hotspots, and the analysis of amelogenin sequences for gender determination. The dodecaplex protocol may be applied to most of the couples requesting PGD for DMD in whom the female partner is a carrier of a deletion. This generic approach will allow prompt response to the PGD referrals by reducing the pre-clinical PGD work-up. It was successfully applied in three DMD families, resulting in the birth of a girl as well as in a healthy ongoing pregnancy.     

荧光原位杂交技术在检测假性肥大型肌营养不良症缺失型携带者中的应用

目的:应用荧光原位杂交(FISH)筛查技术检测假性肥大型肌营养不良症(DMD/BMD)缺失型携带者。方法:以外显子特异Cosmid DNA为探针(含18个外显子),采用中期和间期单色FISH技术,对9例正常男、女性及来自不同缺失型DMD/BMD家系的5例女性外周血标本、来自健康孕妇的2例羊水和2例绒毛标本进行分析。结果:72～100％外周血淋巴细胞中期相或间期核、60～70％羊水细胞间期核、95～99％绒毛细胞间期核显示预期信号。FISH检出1名、排除2名缺失型携带者。结论:充分利用FISH技术优点,结合现有其它技术,可有效筛查DMD/BMD缺失型携带者,并为女性胎儿DMD/BMD缺失型携带者产前诊断奠定基础。     

Dystrophin immunostaining of muscle from Chinese patients with various neuromuscular diseases.

The localization of dystrophin was studied using the immunohistochemical method of diagnostic muscle specimens from 68 patients, aged 9 days to 65 years, with various neuromuscular disorders. Additionally muscle specimens from 2 normal humans and 2 normal mice were used as positive controls, and those from 2 mice with x-linked muscular dystrophy as negative controls. The specimens from all 14 Duchenne muscular dystrophy (DMD) patients, including one with preclinical DMD, showed negative dystrophin staining except for two which had 0.2% to 0.8% positive fibers. The mdx mice also showed negative dystrophin staining. In Becker muscular dystrophy (BMD), muscle fibers stained in a patchy or discontinuous fashion. Two symptomatic DMD carriers exhibited a distinct mosaic pattern of dystrophin positive and negative fibers. In contrast, dystrophin was present in all 7 biopsies from patients with 4 other types of muscular dystrophy (limb-girdle, congenital, myotonic and facioscapulohumeral). Other specimens, those from normal humans and control mice, revealed homogeneous immunostaining along the surface membranes of all muscle fibers. We thus conclude that immunohistochemical dystrophin staining can aid in differentiating DMD from preclinical DMD or BMD, as well as in the detection of DMD carriers.     

Duchenne and Becker muscular dystrophies: genetics, prenatal diagnosis, and future prospects

DMD and BMD are now understood at the genetic, biochemical, and molecular levels. At the genetic level, both disorders result from mutations of the X-linked gene encoding dystrophin. At the biochemical level, DMD results from the deficiency of a large protein called dystrophin, whereas BMD results when dystrophin is present, though abnormal in either amount or molecular structure. To date, thousands of patients have been analyzed for mutations of the dystrophin gene in peripheral blood DNA or alterations of the dystrophin protein in muscle tissue. The severity of the clinical phenotype of these patients has been compared with their dystrophin gene mutations and corresponding dystrophin protein alterations, revealing an unexpectedly high degree of correlation. Thus, information derived from the molecular analysis (DNA or protein) of a particular patient provides a "molecular diagnosis," which is highly predictive of the clinical course that patient can be expected to follow. Because molecular diagnoses are independent of the patient's age, they provide a prognosis for the large majority of muscular dystrophy patients even before clinical symptoms of their disease become apparent. Such prognostic molecular diagnoses have proven particularly valuable when the patient is an isolated case, with no family history for the disorder. Prenatal genetic diagnosis of DMD or BMD may involve use of Southern blot or PCR techniques to search for a deletion in the DNA of at-risk fetuses or more complicated family linkage studies using intragenic and flanking RFLPs. More recently, assay of dystrophin content in fetal skeletal or cardiac muscle from at-risk abortuses has been accomplished, allowing definitive discrimination of affected and normal fetuses in cases in which deletion analyses and family DNA studies were equivocal. In utero fetal skeletal muscle biopsy for dystrophin protein assay has actually been accomplished in at least one at-risk pregnancy in which family DNA studies were uninformative. Dystrophin was present in skeletal muscle from this 20-week-old male fetus, and the pregnancy continued, resulting in the term birth of a healthy male infant. The future holds exciting opportunities for neonatal screening and treatment of these devastating neuromuscular diseases.     

两个肢带型肌营养不良2D型家系SGCA基因遗传分析和产前诊断

目的对中国汉族肢带型肌营养不良2D(limb-girdle muscular dystrophy type 2D,LGMD2D)型的2个家系进行SGCA基因分析,明确病因并在此基础上为该家系中的胎儿进行产前诊断,提供遗传咨询与指导。方法回顾性收集2017年6月至2018年1月在郑州大学第一附属医院就诊的2个LGMD2D型家系,提取先证者和其父母的外周血,通过探针杂交技术对先证者LGMD相关21个基因编码区及其侧翼序列进行高通量测序,进一步采用Sanger测序和/或荧光定量聚合酶链反应对先证者父母目标基因区域进行检测,同时验证变异来源;明确先证者病因后,进一步对家系中胎儿进行产前诊断。结果家系1:先证者存在SGCA基因c.218C>G(p.P73R)和c.101G>A(p.R34H)复合杂合变异;产前诊断结果显示,胎儿与先证者一样同时遗传了父母双方的致病变异,胎儿父母选择终止妊娠。家系2:先证者携带SGCA基因c.218C>T(p.P73L)和该基因第7和第8外显子杂合缺失复合杂合变异,但胎儿不携带上述两变异,家属选择继续妊娠。胎儿足月分娩,随访至2岁,肌酶谱、体格、运动和智力发育均未见异常。结论SGCA基因复合杂合突变是2个LGMD2D型家系的致病原因,其中c.218C>G(p.P73R)、c.218C>T(p.P73L)为尚未报道的新突变。基于高通量测序可快速、准确地对该病进行基因诊断和产前诊断。     

Preimplantation genetic diagnosis (PGD) for Duchenne muscular dystrophy (DMD) by triplex-nested PCR

OBJECTIVES: Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder with an incidence of approximately 1 in 3500 males, caused by mutation in the DMD gene. About 2/3 of DMD cases are caused by gross DMD gene deletion mutations. The purpose of this study was to develop a series of single-cell multiplex-nested PCR protocols for preimplantation genetic diagnosis (PGD) of the most prevalent DMD deletions. METHODS: The protocols were developed on single blood leukocytes from normal males and females and patients with known DMD gene deletion. In the first reaction, 2 of 11 different primer sets (exons 4, 8, 12, 13, 17, 46, 47, 49, 50, 52 and intron 52) were used to allow the simultaneous amplification of different DMD loci and the SRY gender marker, in a single triplex-nested polymerase chain reaction (PCR). Aliquots of this reaction were then subjected to nested PCR in which each locus was amplified individually. Following the successful establishment of single-cell triplex-nested PCR in single leukocytes, the technique was employed in five clinical PGD cases. RESULTS: For each DMD locus, more than 50 single leukocytes from healthy controls and more than 100 single leukocytes from affected individuals with known deletions were analyzed. Amplification efficiency for each tested locus was 98-100%. The false-negative rates for each analysis taken separately was <1%. Taken together, however, the results of the triplex-nested PCR analysis had a false-negative rate of 0%. No contamination was detected in all wash-drop blanks tested. We subsequently performed 18 PGD cycles in 5 DMD carriers. A total of 156 embryos were biopsied and successfully analyzed. Of these, 39 affected embryos were detected and 50 unaffected embryos were transferred (mean = 2.9 +/- 1.1 embryos per cycle). These resulted in three biochemical pregnancies and three clinical pregnancies, all of which have culminated in the birth of normal offspring. CONCLUSION: Triplex-nested PCR using 2 of 11 DMD loci and the SRY gender marker allow PGD for >90% of DMD families with known deletions. These protocols are associated with a high amplification efficiency and accuracy.     

Dystrophin protein and RFLP analysis for fetal diagnosis and carrier confirmation of Duchenne muscular dystrophy

A pregnant woman with indeterminate Duchenne muscular dystrophy (DMD) carrier status, but with DMD diagnosed in her deceased brother (unavailable for study), presented for prenatal diagnosis, intending to continue the pregnancy only if proven unaffected with DMD with near absolute certainty. Creatine kinase (CK) assays to clarify carrier status were inconclusive. Male sex in the fetus was identified, but DNA restriction fragment length polymorphism (RFLP) analysis was not yet available to this centre to investigate the possible transmission of the DMD gene, and the pregnancy was terminated. Tissue histology and dystrophin protein analysis demonstrated the absence of DMD. In a situation with proven maternal carrier status, future fetal inheritance of the opposite maternal X chromosome would indicate the presence of DMD. However, maternal carrier status remained in doubt through a second pregnancy, even with RFLP studies, and was finally established when dystrophin analysis confirmed the presence of DMD in the second fetus. Histologic findings are presented, contrasting features in the two fetuses. The value of dystrophin analysis for establishing the diagnosis of fetal DMD, in this case proving maternal carrier status in a difficult situation, and for demonstrating DMD gene:RFLP haplotype relationships is illustrated.     

Immunohistochemical study of dystrophin in neuromuscular disorders.

Dystrophin, a protein product of the gene that is affected in Duchenne/Becker muscular dystrophy (DMD/BMD), is localized on the sarcolemma of muscle fibers. We tried to study various neuromuscular disorders, including DMD/BMD and their carriers, by the immunohistochemical method with two types of anti-dystrophin antibodies. No dystrophin stain was found on the muscles of cases of DMD. In cases of BMD, partial deficiency or a mosaic appearance of dystrophin was found. In members of DMD/BMD families, polyclonal antibody stains did not show definite membrane abnormality. However, partial deficiency or a mosaic appearance of dystrophin on muscle membranes was found in the carriers by a monoclonal anti C-terminal antibody stain. The explanation may be: 1) more non-specific antigen-antibody cross reactions occurred in the polyclonal antibody stain; and 2) a partial defect exists, such as a segmental deletion of the C-terminal portion of dystrophin. Dystrophin study in muscle diseases is a helpful tool for the following reasons: 1) it improves diagnostic accuracy and helps to differentiate variant types of muscle disorders; 2) it makes an early diagnosis possible before the onset of the symptoms of DMD/BMD; and 3) it detects nonsymptomatic carriers of DMD/BMD. However, without the aid of a genetic study, dystrophin antibody stains cannot absolutely rule out the diagnosis of carriers.     

Prenatal diagnosis of single-gene disorders

Genetic disease can occur due to imbalance of whole chromosomes, smaller chromosome microdeletions or duplications, or at the single-gene level where even a single base change can cause significant disease. This review focuses on the methods available to achieve genetic diagnosis of a fetus in pregnancy, both in the context of a family history of a known disease-causing gene variant and where there is clinical suspicion of a genetic disorder based on ultrasound findings. The indications for rapid trio whole exome sequencing in the prenatal setting will be considered. Until relatively recently, genetic testing of a fetus invariably required invasive procedures to sample fetal tissue, with associated risk of miscarriage. However, non-invasive methods of achieving prenatal diagnosis by sampling fetal DNA present in maternal blood have undergone considerable development. Current applications of invasive and non - invasive prenatal testing are discussed with clinical case studies.     

Prenatal diagnosis of myotonic muscular dystrophy with linked deoxyribonucleic acid probes

Since the localization of the myotonic muscular dystrophy gene, closer deoxyribonucleic acid markers have been discovered. These now facilitate both presymptomatic and prenatal diagnosis of myotonic muscular dystrophy. We report our prenatal diagnosis experience with six cases in five families. Obstetricians are advised to inform their patients with a family history of myotonic muscular dystrophy of these testing opportunities. The fetus of the mother with myotonic muscular dystrophy who remains in utero until term is at considerable risk, as is the mother herself, of serious obstetric complications.