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。     

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。     

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.     

产前诊断除外肌营养不良蛋白基因外显子44移码突变一例

先证者,男,1+岁,临床诊断为杜氏肌营养不良症(Duchenne muscular dystrophy,DMD).该病发生主要原因是编码肌营养不良蛋白(dystrophin)基因的大片段缺失或突变[1].首先采用多重聚合酶链反应技术针对先证者及其父母的肌营养不良蛋白基因26个外显子(包括3、4、6、8、12、1 3、16、17、19、32、34、41、42、43、44、45、46、47、48、49、50、51、52、60、Pm、pb)进行检测,未见上述外显子缺失.进而采用多重连接依赖的探针扩增(multiplex ligation-dependcnt probe amplification,MLPA)法检测肌营养不良蛋白基因79个外显子,均未见缺失或重复.最终经全基因测序发现先证者第44号外显子一处移码突变:NM_004006.1:c.6391_6392delCA(p.Gln2131AsnfsX3),而先证者父母的44号外显子同步测序均未见异常,且此突变未见报道.通过寡核苷酸多态性数据库(www.ncbi.nlm.nih.gov/SNP)、国际人类基因组单体型图计划(www.hapmap,org)和“华大基因千人基因组计划( www.genomics.cn)”中72个中国人样本的数据分析排除多态可能,同时经PDB数据库(www.pdb.org)蛋白功能预测可能造成膜蛋白结构域的改变,因此考虑此突变为新发或生殖腺嵌合造成的致病突变.     

杜氏肌营养不良家系产前诊断一例

本文通过联合应用多重连接依赖性探针扩增(MLPA)、二代测序(NGS)、荧光定量PCR技术和短串联重复序列分析等方法,确诊了1例MLPA结果为阴性而NGS提示杜氏肌营养不良(DMD)基因第19外显子部分区域缺失突变导致的DMD先证者,并对该家系进行了产前诊断,胎儿为突变携带者。提示,DMD家系产前诊断MLPA结果为阴性时需谨慎,多种方法联合的检测及分析可提高DMD基因罕见突变的检出率,为遗传咨询和产前诊断提供依据。     

假肥大型肌营养不良症的基因诊断

假肥大型肌营养不良症(DMD)是一种常见的 X 连锁隐性遗传病。以往都是通过血清肌酸磷酸激酶(CPK)值和家系分析的方法来检出携带者。通过流产所有高危男胎来防止患儿出生。重组 DNA 技术的应用使该病的携带者检出和产前诊断有了较大发展。目前 DMD 致瘸基因已被定位于 X 染色体短臂2区1带(Xp21),并初步分析了 DMD 基因的结构和基因产物。通过限制性片段长度多态(RFLPs)的连锁分析,可以准确地进行 DMD 携带者检出和产前诊断,应用cDNA 探针可以通过检出 DMD 致病基因的部分缺失而准确地检出携带者和产前诊断。     

定量多重PCR在筛本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，应用于携带筛查和产前诊断。运用多重ＰＣＲ技术对１２个ＤＭＤ／ＢＭＤ家系先证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名缺失型患中６名母亲是与     

Duchenne型肌营养不良病高危儿的产前基因诊断

应用ＤＮＡ分子杂交和１８对引物的多重聚梧酶链反应技术，对１０例Ｄｕｃｈｅｎｎｅ型肌营养不良病的高危胎儿进行产前基因诊断，结果：７例男性儿中有３例存在与先证者相同的基因缺失，诊断为ＤＭＤ患儿，均行人工流产术，另４例为正常男性胎儿。３例女性胎儿，１例为突变基因携带者，２例为正常胎儿。以上１０例均经出生后复检证实。提示：ｍＰＣＲ技术简单快速，特异性高，是一种很好的基因缺失筛查方法，本研究还对ＤＭＤ高危胎     

五重巢式PCR同步检测单细胞杜氏肌营养不良基因和性别的研究

目的 建立五重巢式PCR技术同步检测单细胞DMD基因和性别,探讨该技术在杜氏肌营养不良症的植入前诊断（DMD-PGD）的可行性。方法 获取正常男性、女性单个淋巴细胞和无DMD家庭史的单个胚胎细胞,用五重巢式PCR技术检测DMD基因外显子17、19、44、48和SRY基因。结果 在正常男性单个淋巴细胞扩增成功率为96.7%（145/150）,假阳性率为4%（1/25）,假阴性率为0（0/25）。在正常女性单个淋巴细胞扩增成功率为98%（147/150）,假阳性率为0（0/25）,假阴性率为0（0/25）。15个胚胎细胞扩增成功率为100%（66/75）,假阳性率为0（0/25）。结论 本文建立的单细胞DMD基因外显子17、19、44、48和SRY基因五重巢式PCR技术具有较高的敏感性和特异性,有望应用于DMD-PGD。     

进行性肌营养不良患儿肌肉标本金属蛋白酶组织抑制剂 1的表达及临床意义

目的探讨金属蛋白酶组织抑制剂 1(TIMP 1)在进行性肌营养不良(PMD)发病中的作用。 方法中国医科大学附属第一医院等单位于2002年4月至2003年3月，借助免疫组织化学、双免疫荧光标记和Western印迹分析的方法，检测Duchenne型肌营养不良(DMD)、Becker型肌营养不良(BMD)和先天性肌营养不良(CMD)患儿活检肌肉标本中TIMP 1的表达和细胞定位。 结果免疫组织化学和双免疫荧光标记结果显示TIMP 1在正常肌肉的血管内皮细胞处表达；免疫组织化学和Western印迹分析均显示在PMD萎缩的肌肉中TIMP 1的表达明显增强；双免疫荧光标记进一步显示TIMP 1在再生肌肉纤维、巨噬细胞和巨噬细胞浸润的坏死纤维中明显表达，DMD和CMD肌肉中的肌内膜和肌束膜中某些激活的成纤维细胞也强烈表达TIMP 1。 结论在PMD病变部位TIMP 1产生增多及其分布类型提示TIMP 1可能参与了PMD的发病。     

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.     

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.     

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.     

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.     

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.     

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.     

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.