Fluorescent multiplex linkage analysis and carrier detection for Duchenne/Becker muscular dystrophy.

We have developed a fast and accurate PCR-based linkage and carrier detection protocol for families of Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD) patients with or without detectable deletions of the dystrophin gene, using fluorescent PCR products analyzed on an automated sequencer. When a deletion is found in the affected male DMD/BMD patient by standard multiplex PCR, fluorescently labeled primers specific for the deleted and nondeleted exon(s) are used to amplify the DNA of at-risk female relatives by using multiplex PCR at low cycle number (20 cycles). The products are then quantitatively analyzed on an automatic sequencer to determine whether they are heterozygous for the deletion and thus are carriers. As a confirmation of the deletion data, and in cases in which a deletion is not found in the proband, fluorescent multiplex PCR linkage is done by using four previously described polymorphic dinucleotide sequences. The four (CA)n repeats are located throughout the dystrophin gene, making the analysis highly informative and accurate. We present the successful application of this protocol in families who proved refractory to more traditional analyses.     

Deletion of Dystrophin In-Frame Exon 5 Leads to a Severe Phenotype: Guidance for Exon Skipping Strategies

Duchenne and Becker muscular dystrophy severity depends upon the nature and location of the DMD gene lesion and generally correlates with the dystrophin open reading frame. However, there are striking exceptions where an in-frame genomic deletion leads to severe pathology or protein-truncating mutations (nonsense or frame-shifting indels) manifest as mild disease. Exceptions to the dystrophin reading frame rule are usually resolved after molecular diagnosis on muscle RNA. We report a moderate/severe Becker muscular dystrophy patient with an in-frame genomic deletion of DMD exon 5. This mutation has been reported by others as resulting in Duchenne or Intermediate muscular dystrophy, and the loss of this in-frame exon in one patient led to multiple splicing events, including omission of exon 6, that disrupts the open reading frame and is consistent with a severe phenotype. The patient described has a deletion of dystrophin exon 5 that does not compromise recognition of exon 6, and although the deletion does not disrupt the reading frame, his clinical presentation is more severe than would be expected for classical Becker muscular dystrophy. We suggest that the dystrophin isoform lacking the actin-binding sequence encoded by exon 5 is compromised, reflected by the phenotype resulting from induction of this dystrophin isoform in mouse muscle in vivo. Hence, exon skipping to address DMD-causing mutations within DMD exon 5 may not yield an isoform that confers marked clinical benefit. Additional studies will be required to determine whether multi-exon skipping strategies could yield more functional dystrophin isoforms, since some BMD patients with larger in-frame deletions in this region have been reported with mild phenotypes.     

Detection of carriers of deletions in the dystrophin gene in Bulgarian DMD-BMD families

Analysis of Bulgarian Duchenne/Becker muscular dystrophy (DMD/BMD) patients has demonstrated that deletions spanning exon 4 or exon 48 of the dystrophin gene account for about half of all patients, and that female relatives from these families constitute nearly 40% of all patients who require diagnosis of carrier status. We propose a relatively simple and inexpensive assay for the detection of deletion carriers based on a duplex PCR with radioactive 5 end labeling of one of the PCR primers for each exon. The PCR amplification is performed under conditions of exponential relationship be tween template DNA and the amount of PCR product obtained, thus facilitating gene dosage. The quantification of the products, and especially the use of a coefficient estimating of the relative proportion of each exon in the total densitometric area, provide a reliable differentiation between carriers and non-carriers.     

Screening for DMD/BMD deletion carriers by fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) serves as an excellent alternative for direct detection of heterozygous deletions. Using a set of exon-specific cosmid DNA probes representing 18 exons, one-color FISH on metaphase and interphase preparations was performed to identify Duchenne/Becker muscular dystrophy (DMD/BMD) deletion carriers. The peripheral blood samples from 9 normal male or female controls and 5 females of independent DMD/BMD families, as well as 2 amniotic fluid specimens and 2 chorionic villus samples (CVS) from normal pregnant females, were analyzed. Expected signals were displayed in 72-100% of peripheral blood lymphocyte metaphases or interphases, 60-70% of amniocyte interphases, and 95-99% of chorionic villus cell interphases. One suspected female was identified as a deletion carrier and two were excluded. The results indicated that metaphase and interphase FISH were both useful for detection of heterozygous deletions. FISH, in combination with other available techniques, allowed efficient screening of DMD/BMD deletion carriers. The study also offered preliminary results in support of an approach to prenatal diagnosis of potential fetal carriers.     

Molecular diagnosis of Duchenne/Becker muscular dystrophy by polymerase chain reaction and microsatellite analysis

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive genetic disorders resulting from mutations in the dystrophin gene. About two-thirds of the affected patients have large deletions or duplications, which occur in the 5' and central region of the gene. The remaining DMD/BMD cases show no deletions, so they cannot be easily identified by current strategies. In these DMD/BMD families, a linkage analysis that involves DNA markers of the flanking and intragenic dystrophin gene are necessary for carrier and prenatal diagnosis. We analyzed eighteen deletion-prone exons of the gene by a polymerase chain reaction (PCR) in order to characterize the molecular defects of the dystrophin gene in Korean DMD/BMD families. We also performed a linkage analysis to assess the usefulness and application of six short tandem repeat markers for molecular diagnosis in the families. We observed a deletion that eliminated the exon 50. Also, a linkage analysis in the families with six short tandem repeat (STR) markers showed heterozygosity at most of the STR markers. The haplotype analysis was useful for detecting the carrier status. This study will be helpful for a molecular diagnosis of DMD/BMD families in the Korean population.     

Direct carrier detection by in situ suppression hybridization with cosmid clones of the Duchenne/Becker muscular dystrophy locus

Summary A basic problem in genetic counseling of families with Duchenne/Becker muscular dystrophy (DMD/BMD) concerns the carrier status of female relatives of an affected male. In about 60% of these patients, deletions of one or more exons of the dystrophin gene can be identified. These deletions preferentially include exon 45, which can be detected by multiplex polymerase chain reaction (PCR) and Southern blot analysis of genomic cosmid clones that map to this critical region. As a new approach for definitive carrier detection, we have performed chromosomal in situ suppression (CISS) hybridization with these cosmid clones in female relatives of four unrelated patients. In normal females, most metaphases showed signals on both×chromosomes, whereas only one×chromosome was labeled in carriers. Our results demonstrate that CISS hybridization can define the carrier status in female relatives of DMD patients exhibiting a deletion in the dystrophin gene.     

Normal human genomic restriction-fragment patterns and polymorphisms revealed by hybridization with the entire dystrophin cDNA.

Since the complete cDNA for the gene that causes X-linked recessive Duchenne/Becker muscular dystrophy (DMD/BMD) when mutated or deleted has recently been cloned and made generally available, DNA-based diagnostic studies of affected males and their families have entered into a new era. This communication sets forth the standard patterns of restriction fragments that are detected when normal human DNA cleaved with either HindIII or BglII is hybridized with seven contiguous segments comprising the entire 14-kb cDNA. Collectively, the more than 60 restriction fragments allow visualization of approximately 350 (HindIII) to 400 (BglII) kbp. This corresponds to the exon-containing one-fifth of the total genomic length of this gene, including the 3' untranslated region. Twelve two-allele restriction-site polymorphisms that span the entire length of the gene were detected with the cDNA probes and allele frequencies determined. A diagnostic approach is proposed that starts with deletion screening of DNA from male probands, includes carrier detection based on relative fragment intensities, and extends to RFLP detection using the same autoradiographs prepared for deletion screening. Our results on deletion analysis of 32 DMD/BMD families are presented in an accompanying paper.     

Evaluation of point mutations in dystrophin gene in Iranian Duchenne and Becker muscular dystrophy patients: introducing three novel variants

Duchenne and Becker muscular dystrophies (DMD and BMD) are X-linked neuromuscular diseases characterized by progressive muscular weakness and degeneration of skeletal muscles. Approximately two-thirds of the patients have large deletions or duplications in the dystrophin gene and the remaining one-third have point mutations. This study was performed to evaluate point mutations in Iranian DMD/BMD male patients. A total of 29 DNA samples from patients who did not show any large deletion/duplication mutations following multiplex polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification (MLPA) screening were sequenced for detection of point mutations in exons 50–79. Also exon 44 was sequenced in one sample in which a false positive deletion was detected by MLPA method. Cycle sequencing revealed four nonsense, one frameshift and two splice site mutations as well as two missense variants.     

Identification of deletions and duplications of the DMD gene in affected males and carrier females by multiple ligation probe amplification (MLPA)

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused in the majority of cases by deletions of the DMD gene and are readily detectable in affected males by multiplex polymerase chain reaction (PCR). However, different approaches must be used for the identification of female carriers, in which deletions are not detectable by PCR, because of the presence of a normal X chromosome. In this study, we used the multiple ligation probe amplification (MLPA) tool for the identification of female carriers of DMD deletions or duplications in 12 families with a single affected male, 10 of which were previously diagnosed as carriers of a DMD rearrangement, and the remaining two as having an unknown disease-causing mutation. In all the investigated affected males, MLPA analysis confirmed the presence of a DMD rearrangement, and in six of them allowed the refinement of the breakpoints. In 12 female relatives of the affected patients, MLPA analysis showed a DMD deletion or duplication, confirming their carrier status. Two of these were the mother and the sister of a patient whose disease-causing mutation was not known. MLPA analysis thus proved to be an useful tool for the analysis of both affected males and females carriers of DMD rearrangements in cases in which the disease-causing mutation in the affected male was not known, providing useful information for the genetic counselling of the family.Valentina Gatta and Oronzo Scarciolla contributed equally to this work.     

A small deletion in the Duchenne/Becker muscular dystrophy locus —a functionally important region?

Summary A DNA deletion in a patient with Becker muscular dystrophy (BMD) has been delineated by restriction endonuclease mapping. The deletion is unusually small, removing six kilobases (kb) of DNA distal to pERT 87-1 (DXS164). This region has previously been shown to contain an exon of a candidate gene which, when defective, causes Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy. Removal of this exon and surrounding DNA is apparently sufficient, in this case, to cause a BMD phenotype. The occurrence of this deletion in DXS164 would appear to confirm that this region is part of the BMD locus. Many DMD patients have deletions in and around this region, adding further evidence for the allelic nature of the two disorders. This fortuitous deletion may identify a functionally important domain of the protein product in terms of the severity of phenotype manifested.     

多重连接探针扩增方法在假肥大性肌营养不良产前基因诊断中的应用

假肥大性肌营养不良(Duchenne/Becker muscular dystrophy, DMD/BMD)是一种由于DMD基因突变导致的X连锁隐性致死性遗传病。目前没有有效的治疗方法。为建立一种既可以对携带者进行检测又可以进行产前基因诊断的方法, 文章联合应用多重连接探针扩增技术(Multiplex ligation-dependent probe amplification, MLPA)和短串联重复序列(Short tandem repeats , STR)为遗传标记连锁分析的方法对26例有高风险再生育患儿的假肥大性肌营养不良家系的孕妇通过羊水穿刺进行产前基因诊断。26例进行产前基因诊断的羊水标本中有7例诊断为男性患儿, 4例诊断为女性携带者。MLPA可以作为筛查DMD基因缺失和重复突变的首选方法。联合应用MLPA和STR连锁分析, 可以提高假肥大性肌营养不良的产前基因诊断率。     

Further studies of gene deletions that cause Duchenne and Becker muscular dystrophies

Fetal muscle cDNA clones covering at least 11.4 kb of the Duchenne muscular dystrophy (DMD) gene sequence were used to identify a deletion-prone region in DNA from DMD and Becker muscular dystrophy (BMD) patients. Of 36 BMD cases, 17 (47%) had deletions and all of the deletions began in the same intron of the gene. Of 107 DMD patients, 27 (25%) were deleted for this region, and 19 deletions originate in the same intron. Using a cDNA probe for an adjacent region of the gene, 32 new deletions were detected in DMD patients (total 44%). No new BMD deletions were detected. The DMD deletions were very heterogeneous. Thus two cDNA probes covering 2.4 kb could detect 53% of these deletions. Considering the whole locus, DMD and BMD are caused by a deletion of the gene sequence in at least 67% of cases.     

Molecular diagnosis of duchenne muscular dystrophy: past, present and future in relation to implementing therapies

Duchenne muscular dystrophy (DMD) is the commonest and best-known of the muscular dystrophies. Being an X-linked disorder, it affects mainly boys. The disease gene was identified in 1987, with the majority of mutations demonstrated to be large-scale deletions. Current best practice molecular diagnosis includes multiplex ligation-dependent probe amplification (MLPA) followed by direct sequencing of all exons at the genomic level, or from cDNA, in order to detect point and other small mutations. The difference between DMD and the allelic Becker muscular dystrophy (BMD) is whether the precise mutation in the gene is a null mutation or results in a modified still partially functional protein. Over the last few years, significant progress has been made in moving experimental therapies into clinical trials, with one of the most promising possible therapies being anti-sense oligonucleotide induced exon-skipping, which converts DMD to BMD. In order to maximise the benefit from future therapies, it will be necessary to start administering the therapies as early as possible in the life of the affected boys, before significant muscle loss occurs. This will require early diagnosis, which evidence suggests is best achieved through population screening. Population screening also allows the avoidance of multiple affected boys in families with no previous family history.     

Canine X-linked muscular dystrophy in Japan (CXMDJ).

The purpose of this study was to develop a strain of canine X-linked muscular dystrophy (CXMD), a model of Duchenne muscular dystrophy, in Japan. A female beagle was artificially inseminated with frozen-thawed spermatozoa derived from an affected golden retriever. Subsequently, two carrier female dogs (G1 carriers) and four normal male littermates were produced. Thereafter, the two G1 carriers were mated with beagle sires. As a result, each bitch whelped three times, and out of 54 pups, 17 affected male descendants, and 11 carrier female descendants (G2 carriers) were detected. One G2 carrier was then mated with a beagle sire and 15 pups in two whelpings were produced, including five affected males and four carrier females (G3 carriers). A total of 10 female beagles were artificially inseminated to evaluate the fertility of the frozen-thawed spermatozoa from the two affected dogs. The whelping rates of the two affected dogs were 4/5 and the litter sizes were 5.0 +/- 1.41 and 6.0 +/- 0.82, respectively. These results indicate that a canine X-linked muscular dystrophy colony has been established in Japan. We called them CXMDJ.     

正常中国人和DMD患者X染色体Xp~(21)区的RFLP研究

本文应用从人类X柒色体Xp~(21)区不同部位分离得到的9种DNA探针,分析了100名正常中国人,38名DMD患者及其母亲X柒色体Xp~(21)区的14个限制性位点多态性(RSP;又称限制性片段长度多态性,RFLP)。发现正常的X染色体与携带DMD基因的X染色体Xp~(21)区的RFLP频率没有明显差别;在38例DMD患者中有7例的X染色体有DNA片段缺失;在本文分析的24例患者母杀中有17例是DMD基因携带者,她们在Xp~(21)区的RFLP均存在杂合的多态性,因此可以应用RFLP连锁分析对这些家系进行DMD的产前诊断。     

Brother/sister pairs affected with early-onset, progressive muscular dystrophy: molecular studies reveal etiologic heterogeneity.

An autosomal recessive (AR) form of muscular dystrophy that clinically resembles Duchenne/Becker types exists, but its frequency is unknown. We have studied three unrelated affected brother/sister pairs and their families for deletions and polymorphisms with the entire dystrophin cDNA and other DNA probes from the Xp21 region to test for involvement of the DMD locus. In family 1 a large intragenic deletion was found in the affected male. The affected sister was heterozygous for this deletion, but the mother was not, implying germinal mosaicism. In family 2, no deletion was detected in the affected male. RFLP analysis revealed that the affected male and an unaffected sister shared a complete Xp21 haplotype while the affected sister had inherited a recombinant Xp21 region resulting from a crossover between pERT 87-15 and J-Bir. Only the 5' region of the dystrophin gene was shared with the affected boy. X-inactivation studies using a polymorphism in the 5'-flanking region of the HPRT gene, in conjunction with methylation-sensitive enzymes, revealed random X inactivation in the affected girl's leukocytes. In a muscle biopsy from the affected male, the dystrophin protein was present in normal amount and size. Family 3 was informative for four RFLPs detected with dystrophin cDNA probes which span the entire gene. The affected male was found to share the complete dystrophin RFLP haplotype with his unaffected brother, while his affected sister had inherited the other maternal haplotype. It is concluded that the clinical presentation of early-onset, progressive muscular dystrophy in a male and in his karyotypically normal sister can be caused by mutations at different loci. While in family 1 a deletion in the dystrophin gene is responsible, this gene does not appear to be involved in families 2 and 3.     

RFLPs for Duchenne muscular dystrophy cDNA clones 9 and 10.

The complete 14-kb cDNA for the gene causing the X-linked recessive muscular dystrophy (MD) type Duchenne (DMD) and Becker (BMD) has recently been cloned and made available for deletion/duplication screening in patients. It detects 65 exon-containing nonpolymorphic HindIII fragments spread over a gene locus of about 2,000 kb. When the entire DMD cDNA is used, deletions/duplications can be found in about 65%-70% of affected patients, permitting direct carrier detection by densitometric scanning. But in cases where no deletion/duplication is detectable, RFLP analysis, specially favored within the gene, will be the method for carrier-status determination. Clones 9 and 10-1.2-kb and 0.7-kb fragments, respectively, of the 14-kb DMD cDNA--have been hybridized with human genomic DNA digested by nine different restriction enzymes. Five RFLPs, involving Asp700, PvuII, XbaI, and EcoRV sites, were detected, and Mendelian inheritance could be demonstrated. Since clones 9 and 10 are localized telomeric to the mutation-hot-spot region, their polymorphisms are thought to be very helpful as flanking markers for indirect carrier detection in families with a family history of DMD/BMD. Moreover, these RFLPs can be used for direct carrier detection or exclusion in families with patients showing a deletion/duplication in the region of p9 or p10.