Duchenne型肌营养不良症家系的多重PCR和STR—PCR基因诊断

目的该研究率先开展温州地区Duchenne型肌营养不良症（DMD）家系的缺失基因诊断特别是STR单体型连锁基因诊断,为基于DMD症状前、携带者基因诊断结果的遗传咨询和生育指导提供依据。方法针对4例DMD先证者,采用多重PCR检测常见18个外显子缺失,进行直接基因诊断。针对未能发现常见外显子缺失的DMD先证者及其有关家系成员,采用短串联重复序列（STR）PCR检测5个位点（3’CA、44CA、45CA、49CA和50CA）STR多态性,进行间接单体型连锁基因诊断。结果家系二的先证者缺失外显子3、4和6。其余3个家系的先证者的异常x染色体均肯定来源于其母亲。家系一先证者外婆肯定是携带者。家系三先证者年幼（4周岁）弟弟肯定为正常人,将来年龄大了也不会发病,先证者外婆肯定是携带者。家系四先证者刚出生的妹妹肯定是遗传携带者,将来其生育儿子有遗传患病风险。结论该研究的DMD家系的缺失和STR单体型连锁基因诊断,特别是对症状前男孩的诊断、对无患病后代的女性携带者的检出,具有非常重要的实际意义,可以为遗传咨询和生育指导提供可靠依据。     

Direct method for prenatal diagnosis and carrier detection in Duchenne/Becker muscular dystrophy using the entire dystrophin cDNA

DNA sequence polymorphisms (RFLPs) have been widely used as genetic markers for identification of the X chromosome that carries the mutation for Duchenne muscular dystrophy (DMD) in affected families, but serious limitations and pitfalls are associated with this approach [Darras et al., 1987]. The complementary DNA (cDNA) of the DMD gene has recently been isolated and shown to detect partial gene deletions in a large proportion of patients [Koenig et al., 1987]. Two prenatal studies are presented to illustrate how the unambiguous identification of deletion mutations by cDNA probes permits direct DNA-based diagnoses with high accuracy and in otherwise uninformative families. In a single proband family, DNA marker analysis had determined that the Xp21 chromosomal region present in the affected male was also carried by a male fetus in a subsequent pregnancy. Analysis of this family's DNA with probes covering the entire 14 kb cDNA revealed a small deletion in the affected male that was not present in the fetus nor in the mother. In the second family the fetus was a female deletion carrier identified by comparing intensities of restriction fragments. Since 1/3 of all DMD patients are thought to result from new mutations and most families have only single affected males, the cloned cDNA probes now available are likely to revolutionize DNA-based diagnostic studies in this disorder. More reliable, more rapid and less expensive than linkage studies with DNA polymorphisms, this method will be informative in the more than 50% of DMD/BMD cases that have deletion mutations.     

Identification of female carriers for Duchenne and Becker muscular dystrophies using a FISH-based approach

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive neuromuscular diseases caused by dystrophin gene mutations. Deletions, or more rarely duplications, of single or multiple exons within the dystrophin gene can be detected by current molecular methods in approximately 65% of DMD patients. Mothers of affected males have a two-thirds chance of carrying a dystrophin mutation, whilst approximately one-third of affected males have de novo mutations. Currently, Southern blot analysis and multiplex PCR directed against exons in deletion hot spots are used to determine female carrier status. However, both of these assays depend on dosage assessment to accurately identify carriers since, in females, the normal X chromosome is also present. To obviate quantitation of gene dosage, we have developed exon-specific probes from the dystrophin gene and applied them to a screen for potential carrier females using fluorescence in situ hybridization (FISH). Cosmid clones, representing 16 exons, were identified and used in FISH analysis of DMD/BMD families. Our preliminary work has identified multiple, informative probes for several families with dystrophin deletions and has shown that a FISH-based assay can be an effective and direct method for establishing the DMD/BMD carrier status of females.     

Possibilities and limitation of prenatal diagnosis and carrier determination for Duchenne and Becker muscular dystrophy using cDNA probes.

Two cDNA probes, cf23a and cf56a, identify deletions of selected exons in about 50% of our DMD/BMD patients. We have estimated the most likely order of the 11 exons detectable with both probes with respect to the different extensions of the deletions. In one of our BMD pedigrees, the observed deletion could be traced in the affected males through three generations. This result shows that with the use of cDNA probes detecting deletions, the only risk of error in genomic prenatal diagnosis is the general high frequency of new mutations for DMD/BMD. This is important progress in diagnosis compared to the 2 to 5% risk of misdiagnosis because of crossing over events using conventional linkage analysis with bridging or intragenic probes. The first prenatal diagnosis of an unaffected fetus of a woman who is a DMD carrier according to ultrasound examination is described. In one of our DMD males, the cDNA probe cf56a detects a deletion breakpoint. His sister also shows the altered band and is therefore a DMD carrier, while his mother has a totally normal band pattern. The interpretation of this observation could be either germline mosaicism or two identical new mutations. The identification of deletion breakpoints is a new diagnostic strategy, especially for carrier determination, which excludes misdiagnosis owing to crossing over events and the problems of dosage estimation. It is, however, limited by the low frequency of breakpoints detectable with cDNA probes. Therefore, the generation of new intron probes in this region is an important goal.     

High frequency of de novo deletions in Mexican Duchenne and Becker muscular dystrophy patients. Implications for genetic counseling

Duchenne muscular dystrophy (DMD) is the most common lethal hereditary neuromuscular disease. As there is no effective treatment, accurate carrier detection is essential for genetic counseling and prevention. Although linkage analysis has been widely used for this purpose, being an indirect analysis it has several limitations. Using linkage analysis for carrier detection, we found serious limitations, mainly because 82.9% of all proposita were isolated cases. We used quantitative polymerase chain reaction for direct carrier detection in families with exon deletions and found a higher than expected frequency of de novo deletions (62.2%). Furthermore, only 20.7% of the mothers of isolated deletion DMD/Becker muscular dystrophy (BMD) patients were found to be carriers. This result suggests that the Mexican population has a high frequency of de novo DMD mutations.     

Preimplantation diagnosis of non-deletion Duchenne muscular dystrophy (DMD) by linkage polymerase chain reaction analysis

The use of preimplantation diagnosis for sex determination and detection of exon deletion means that unaffected babies can be born to parents suffering from Duchenne muscular dystrophy (DMD). However, those who do not have exon deletion should also be considered for further investigation. A new method, known as linkage analysis, has been developed to diagnose the presence of non-deletion DMD in preimplantation embryos. Linkage analysis uses informative intragenic and flanking markers to track the chromosome bearing the mutated gene. The present study reports the analysis of two polymorphic sites, in blastomeres biopsied from embryos from a female carrier of DMD. A single male embryo was obtained who had inherited alternate maternal alleles to the woman's affected surviving son, and this embryo was transferred.      

A grandpaternally derived de novo deletion within Xp21 initially presenting in carrier females diagnosed as Kugelberg-Welander syndrome

We report on two sisters with a history of muscle weakness and an electromyogram (EMG) diagnosis of Kugelberg-Welander syndrome (KWS) or juvenile spinal muscular atrophy. A half-brother to these women was diagnosed to have Duchenne muscular dystrophy (DMD). Using molecular probes, we identified a deletion within Xp21 in this isolated case of DMD. Sequences detected by pXJ1.1 are deleted, while fragments detected by pERT87 are intact. Both of these probes are derived from the DMD locus. We have shown that the affected sisters share with their half-brother DNA markers that are linked to the DMD gene and inherited from their maternal grandfather. Dosage analysis of Southern blots show monosomy for pXJ1.1, which has allowed us to determine carrier status within this family and to show that the half-sisters are manifesting DMD carriers.     

Mental retardation locus in Xp21 chromosome microdeletion

Xp21 microdeletion syndrome is associated with variable size Xp21 deletions that usually include the glycerol kinase locus. The clinical phenotypes we studied in this chromosome region include: Xpter – Åland Island eye disease (AIED) -adrenal hypoplasia (AH) -glycerol kinase (GKD) -Duchenne muscular dystrophy (DMD) -retinitis pigmentosa (RP) -ornithine transcarbamylase (OTC) -centromere. In a compilation of 18 individuals in 14 families with the AH, GKD, and DMD loci deleted, 17 were male and all were developmentally delayed. In contrast, we report mentally retarded female carriers in two Xp21 deletion syndrome families with DMD, GKD, and AH in affected males. In the first family with normal karyotypes, a submicroscopic deletion was associated with DMD in the retarded male and with retardation in carrier females. In the second family an X chromosome with a cytogenetically deleted Xp21 distal to the OTC and RP genes segregated in the affected male and retarded female carriers. DNA analysis at the DMD locus verified the cytogenetic findings. This report of mental retardation in otherwise asymptomatic female carriers of Xp21 deletion classifies one form of mental retardation in females. © 1993 Wiley-Liss, Inc.     

PGD for dystrophin gene deletions using fluorescence in situ hybridization

Duchenne muscular dystrophy and Becker muscular dystrophy (DMD and BMD) are caused by mutations in the dystrophin gene (Xp21). In two-thirds of DMD/BMD cases, the mutation is a large deletion of one or several exons. We have established PGD for DMD/BMD using interphase fluorescence in situ hybridization (FISH) analysis on single nuclei from blastomeres for the detection of deletions of specific exons in the dystrophin gene. We performed PGD for two carrier females; one had a deletion of exons 45-50 (DMD), and the other had a deletion of exons 45-48 (BMD). An exon 45-specific probe was used in combination with probes for the X and Y centromeres. Using this straightforward approach, we can distinguish affected and unaffected male embryos as well as carrier female and normal female embryos. Three cycles were performed for each patient, which resulted in a pregnancy and the birth of a healthy girl. To the best of our knowledge, this approach for PGD has not been previously reported. The use of interphase FISH is an attractive alternative to sexing or PCR-based mutation detection for PGD patients with known deletions of the dystrophin gene.     

Alternative splicing of dystrophin mRNA complicates carrier determination: report of a DMD family.

Carrier determination is important for genetic counselling in DMD/BMD families. The detection of altered PCR amplified dystrophin mRNA fragments owing to deletions, insertions, or point mutations has increased the possibilities of carrier determination. However, problems may occur because of alternative splicing events. Here we present a family with a DMD patient characterised by a deletion of exons 45 to 54. At the mRNA level we detected a corresponding altered fragment which served for carrier determination. The mother and the sister of the patient showed the same altered dystrophin mRNA fragment as the patient and are therefore carriers. In the mother two additional altered dystrophin mRNA fragments were detectable, obviously resulting from alternative splicing in the normal allele. The grandmother and two other related females of the patient possess only the normal mRNA fragment. In a further female we detected an altered fragment owing to an mRNA deletion of exon 44. This fragment is created either by alternative splicing or a new mutation. Therefore, the carrier status of this female is still ambiguous indicating problems in carrier determination by the method of dystrophin mRNA analysis.     

定量PCR结合STR多态连锁分析诊断缺失型DMD基因携带者

目的：直接定量PCR结合STR单体型连锁分析，以便更准确检出缺失型家系中DMD基因携带者。方法：在内标引物参照下，PCR扩增22个循环，产物在琼脂糖凝胶分离，EB显色，照相后，扫描定量，同时应用5个STR多态位点的单体型连锁分析，检测缺失型的DMD基因女性携带者。结果：直接定量PCR分析了6个家系中的8个外显子48缺失的DMD母亲或女性同胞，检出7个DMD为携带者；STR多态单体型连锁同样分析这6个家系中的8个女性亲属，亦检出7个DMD基因携带者，二种方法所得结果完全一致。结论：STR多态连锁分析与直接定量PCR法结合，可更准确，全面检出DMD基因携带者。     

应用多个微卫星DNA位点进行Duchenne/ Becker肌营养不良症携带者的检测

目的　筛查和确定 Duchenne/ Becker肌营养不良症 ( Duchenne/ Becker muscular dystrophy,DMD/ BMD)家系的女性成员中的致病基因携带者与正常者 ,为进一步行产前诊断或植入前遗传学诊断提供信息。方法　用 PCR方法对 dystrophin基因第 4 4、4 5、4 9和 5 0内含子以及 5′DMD 的短串联重复序列( short tandem repeats,STR)扩增 ,然后进行基因扫描、软件分析 ,对 4个 DMD/ BMD家系中 2 7个成员的这 5个微卫星 DNA位点的多态性进行连锁分析。结果　在 4个家系 17名女性成员中 (有 1例女性 DMD患者 ) ,系谱和 STR多态性分析结果均符合的 DMD基因肯定携带者有 6名 ;单纯根据 STR多态性连锁分析结果确诊为 DMD基因携带者的女性成员有 5名 ,确诊为正常女性成员有 5名。在这 5个 STR位点中 ,最具多态性的位点是 STR- 4 9,多态性最少的位点是 STR- 5 0。结论　短串联重复序列多态性结合基因扫描能快速、准确、客观地检出 Duchenne/ Becker型肌营养不良症的女性携带者     

Fabry disease: Comparison of enzymatic,linkage, and mutation analysis for carrier detection in a family with a novel mutation (30delG)

Fabry disease (FD) is an X-linked recessive disorder caused by the deficient activity of the lysosomal enzyme α-galactosidase A (α-Gal A). Affected males are reliably diagnosed by demonstration of deficient α-Gal A activity in plasma or leukocytes. However, identification of female carriers is problematic due to Lyonization, requiring mutation identification and/or linkage studies for accurate carrier detection. Here, we describe a large Brazilian kindred with Fabry disease that permitted comparison of biochemical and molecular diagnostic techniques. Initially, the plasma α-Gal A activities were determined in at-risk affected males and potential female carriers; affected males were readily diagnosed, while the females had variable results. To detect carrier females, haplotype analysis using 10 polymorphic markers adjacent to the gene was performed. Subsequently, solid-phase direct sequencing of the α-Gal A gene demonstrated a novel single base deletion in exon 1 (30delG). Discrepancies were observed between the enzymatic and molecular diagnoses in two at-risk females. These findings emphasize the need for precise heterozygote diagnosis by mutation and/or haplotype analyses in all families with Fabry disease. Am. J. Med. Genet. 84:420–424, 1999. © 1999 Wiley-Liss, Inc.     

内含子或外显子缺失DMD家系女性携带者的基因诊断

目的对内含子和(或)外显子缺失的Duchenne型肌营养不良症(Duchenne muscular dystrophy,DMD)家系女性成员进行致病基因携带者的诊断,为进一步行产前诊断或植入前遗传学诊断提供准确的信息。方法利用dystrophin基因5个微卫星位点(STR-44、45、49、40、5′DysⅡ)的多态性连锁分析,同时结合半定量PCR对1个内含子和外显子均缺失的DMD家系(家系5)和1个仅内含子缺失的DMD家系(家系4)的女性成员进行携带者基因诊断。结果家系5的Ⅱ2的STR-50位点基因型为245/245,不是DMD基因携带者。家系4的Ⅱ6、Ⅱ8的STR-45位点基因型为del/172,Ⅲ19为del/178,均为DMD基因携带者。结论STR-PCR多态性分析结合内含子半定量PCR可获得更多诊断信息,更准确地检出内含子缺失的女性携带者,提高诊断率。     

Carrier detection of Duchenne/Becker muscular dystrophy by analysis of STRs loci linked to the gene of dystrophin in Venezuelan families

The Duchenne/Becker Muscular Dystrophy (DMD/BMD) is an X linked recessive lethal disease. The female carrier will transmit the disease gene to half of her sons and half of her daughters; half of the daughters will be carriers, while half will be normal. Half of the sons will be normal and, on average, half will have the disease. It is of particular relevance to be able to detect carrier status among female relatives of the patients for genetic counseling and prenatal diagnosis. The method of Short Tandem Repeat (STR) sequence polymorphism analysis can determine haplotype at normal status or at risk status and, to establish genetic linkage between the mutated gene and the segregated haplotype. We have analyzed 105 members from 15 unrelated Venezuelan families with one or more siblings affected with DMD/DMB and 7 unrelated males. Of the 105, 37 were male (26 affected and 11 normal) and 68 were female. STR sequences (STR44, STR45, STR49, STR50, STR3'DYS) of the gene of the Dystrophin were amplified by polymerase chain reaction (PCR) to analyze allelic polymorphism in the families. Five of the 15 families (33%) had a deletion of one or several of the exons. Of the 68 females, 27 (39.7%) were carriers, 27 (39.7%) were non-carriers and in 14 cases (20.58%) it was not possible to reach a definitive diagnosis. The definitive diagnosis could be established in 79% of the females. This analysis also shows that the mutation occurred on the grandpaternal X chromosome in one family. Hemizygocity was detected and carrier status ascertained in the mother of other patient and in one family we were able to do prenatal diagnosis. The germinal mosaicism could not be excluded in 3 patients.     

Detection of dystrophin deletion carriers using FISH analysis

The detection of carrier status in female relatives of Duchenne/Becker muscular dystrophy patients is not always possible and this poses a problem in genetic counseling. We have developed a simple method that can be used in families in which affected males are characterized by the presence of a deletion within the dystrophin gene. PCR fragments, corresponding to the deleted regions are used as fluorescent probes for hybridization of peripheral lymphocytes nuclei of female relatives. The results obtained clearly demonstrate the feasibility of this method for detecting female DMD/BMD carriers.     

STR-PCR联合SRY-PCR方法在DMD点突变家系产前基因诊断中的应用

目的对点突变型假肥大型肌营养不良症（Duchenne＇s muscular dystrophy,DMD）患者的家系采用STR-PCR联合SRY-PCR分析方法进行产前基因诊断。方法采用STR-PCR连锁分析方法和SRY-PCR方法对20个点突变型DMD家系进行产前基因诊断。结果男性胎儿40%（8/20）,其中患胎62.5%（5/8）;女性胎儿60%（12/20）,其中携带者58.3%（7/12）。结论 STR-PCR连锁分析方法结合SRY-PCR方法用于点突变型DMD家系的产前诊断,是目前一种快速、简便、准确和可行的方法。     

Fabry disease: comparison of enzymatic, linkage, and mutation analysis for carrier detection in a family with a novel mutation (30delG).

Fabry disease (FD) is an X-linked recessive disorder caused by the deficient activity of the lysosomal enzyme alpha-galactosidase A (alpha-Gal A). Affected males are reliably diagnosed by demonstration of deficient alpha-Gal A activity in plasma or leukocytes. However, identification of female carriers is problematic due to Lyonization, requiring mutation identification and/or linkage studies for accurate carrier detection. Here, we describe a large Brazilian kindred with Fabry disease that permitted comparison of biochemical and molecular diagnostic techniques. Initially, the plasma alpha-Gal A activities were determined in at-risk affected males and potential female carriers; affected males were readily diagnosed, while the females had variable results. To detect carrier females, haplotype analysis using 10 polymorphic markers adjacent to the gene was performed. Subsequently, solid-phase direct sequencing of the alpha-Gal A gene demonstrated a novel single base deletion in exon 1 (30delG). Discrepancies were observed between the enzymatic and molecular diagnoses in two at-risk females. These findings emphasize the need for precise heterozygote diagnosis by mutation and/or haplotype analyses in all families with Fabry disease.     

Deletion analysis of DMD/BMD families from the German Democratic Republic and selected regions of Czechoslovakia and Hungary.

Over the last two years we have screened 183 DMD/BMD families requesting prenatal diagnosis. Using cDNA probes cf56a,b we have detected exon deletions in 72 of them. In 62 cases the deletion was also detectable with currently available PCR primers. Deletion analysis for exons 8, 17, and 19, using either PCR or Southern blotting techniques, was performed for 65 of the 111 families which showed no deletions with cf56a,b. Eight of them were deleted for one or more of these exons. PCR offers new possibilities for deletion analysis in families without a living patient using either Guthrie papers or histologically conserved material from the dead patient. In 20 of 25 patients, we observed concordance between the clinical picture and the molecular deletion analysis in accordance with the open reading frame hypothesis. Five patients, however, presented with DMD in spite of our analysis showing an in frame deletion. Carrier determination in families in which DMD is caused by a deletion using linkage, dosage, or breakpoint analysis is discussed.