Uniparental disomy of the entire X chromosome in a female with Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe, progressive, X-linked muscle-wasting disorder with an incidence of approximately 1/3,500 male births. Females are also affected, in rare instances. The manifestation of mild to severe symptoms in female carriers of dystrophin mutations is often the result of the preferential inactivation of the X chromosome carrying the normal dystrophin gene. The severity of the symptoms is dependent on the proportion of cells that have inactivated the normal X chromosome. A skewed pattern of X inactivation is also responsible for the clinical manifestation of DMD in females carrying X;autosome translocations, which disrupt the dystrophin gene. DMD may also be observed in females with Turner syndrome (45,X), if the remaining X chromosome carries a DMD mutation. We report here the case of a karyotypically normal female affected with DMD as a result of homozygosity for a deletion of exon 50 of the dystrophin gene. PCR analysis of microsatellite markers spanning the length of the X chromosome demonstrated that homozygosity for the dystrophin gene mutation was caused by maternal isodisomy for the entire X chromosome. This finding demonstrates that uniparental isodisomy of the X chromosome is an additional mechanism for the expression of X-linked recessive disorders. The proband's clinical presentation is consistent with the absence of imprinted genes (i.e., genes that are selectively expressed based on the parent of origin) on the X chromosome.     

Muskeldystrophien Duchenne und Becker

Duchenne muscular dystrophy (DMD) is the most frequent muscular disorder in infancy. The inheritance is X-linked recessive with mutations in the dystrophin gene (about 65% deletions, about 7% duplications, about 26% point mutations, and about 2% unknown mutations). The genetic model is complex. The sex ratio of the mutations is unequal. Point mutations and duplications arise in spermatogenesis, whereas deletions arise in oogenesis. About 33% of all patients are new mutations; however, most new mutations are germline mosaic. Becker muscular dystrophy is allelic to DMD.     

Direct deletion analysis in two Duchenne muscular dystrophy symptomatic females using polymorphic dinucleotide (CA)n loci within the dystrophin gene

Duchenne muscular dystrophy (DMD) is the most common hereditary neuromuscular disease. It is inherited as an X-linked recessive trait in which males show clinical manifestations. In some rare cases, the disease can also be manifested in females. The aim of the present study was to determine the molecular alteration in two cases of nonrelated DMD symptomatic carriers with no previous history of DMD. Multiplex PCR is commonly used to search for deletion in the DMD gene of affected males. This method could not be used in females because the normal X chromosome masks the deletion of the mutated one. Therefore, we used a set of seven highly polymorphic dinucleotide (CA)(n) repeat markers that lie within the human dystrophin gene. The deletions were evidenced by hemizygosity of the loci under study. We localized a deletion in the locus 7A (intron 7) on the maternal X chromosome in one case, and a deletion in the region of introns 49 and 50 on the paternal X chromosome in the other. The use of microsatellite genotyping within the DMD gene enables the detection of the mutant allele in female carriers. It is also a useful method to provide DMD families with more accurate genetic counseling.     

X-chromosome methylation in manifesting and healthy carriers of dystrophinopathies: concordance of activation ratios among first degree female relatives and skewed inactivation as cause of the affected phenotypes

The X-chromosome activity states of 11 manifesting carriers of dystrophinopathies, all with normal karyotypes, were estimated by restriction fragment length polymorphism (RFLP)-methylation analysis with the probes M27 (DXS255), p2-19(DXS605) and pSPT/PGK (PGK1) to test the role of skewed X-inactivation ratios as the cause of their affected phenotypes. In eight cases preferential inactivation of the putative X chromosome carrying the normal dystrophin allele in 90% of their peripheral lymphocytes was observed, two cases showed non-appparent deviant ratios (6040 and 7030) from the theoretically expected values around the mean of 50% and in one case the three markers employed yielded no information. The analysis of the X-inactivation ratio in six mother-daughter pairs, all non-manifesting Duchenne muscular dystrophy (DMD) carriers, and in the close female relatives of the patients showed: (a) neither of the two X chromosomes was preferentially inactivated with respect to their parental origin; (b) a high concordance among the activation ratios of mothers and daughters, a result difficult to explain just in terms of random X-chromosome inactivation.     

Skewed X inactivation in a female MZ twin results in Duchenne muscular dystrophy.

One of female MZ twins presented with muscular dystrophy. Physical examination, creatine phosphokinase levels, and muscle biopsy were consistent with Duchenne muscular dystrophy (DMD). However, because of her sex she was diagnosed as having limb-girdle muscular dystrophy. With cDNA probes to the DMD gene, a gene deletion was detected in the twins and their mother. The de novo mutation which arose in the mother was shown by novel junction fragments generated by HindIII, PstI, or TaqI when probed with cDNA8. Additional evidence of a large gene deletion was given by novel SfiI junction fragments detected by probes p20, J-Bir, and J-66 on pulsed-field gel electrophoresis (PFGE). Immunoblot analysis of muscle from the affected twin showed dystrophin of normal size but of reduced amount. Immunofluorescent visualization of dystrophin revealed foci of dystrophin-positive fibers adjacent to foci of dystrophin-negative fibers. These data indicate that the affected twin is a manifesting carrier of an abnormal DMD gene, her myopathy being a direct result of underexpression of dystrophin. Cytogenetic analysis revealed normal karyotypes, eliminating the possibility of a translocation affecting DMD gene function. Both linkage analysis and DNA fingerprint analysis revealed that each twin has two different X chromosomes, eliminating the possibility of uniparental disomy as a mechanism for DMD expression. On the basis of methylation differences of the paternal and maternal X chromosomes in these MZ twins, we propose uneven lyonization (X chromosome inactivation) as the underlying mechanism for disease expression in the affected female.     

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.     

X chromosome inactivation in carriers of Barth syndrome.

Barth syndrome (BTHS) is a rare X-linked recessive disorder characterized by cardiac and skeletal myopathy, neutropenia, and short stature. A gene for BTHS, G4.5, was recently cloned and encodes several novel proteins, named "tafazzins." Unique mutations have been found. No correlation between the location or type of mutation and the phenotype of BTHS has been found. Female carriers of BTHS seem to be healthy. This could be due to a selection against cells that have the mutant allele on the active X chromosome. We therefore analyzed X chromosome inactivation in 16 obligate carriers of BTHS, from six families, using PCR in the androgen-receptor locus. An extremely skewed X-inactivation pattern (>=95:5), not found in 148 female controls, was found in six carriers. The skewed pattern in two carriers from one family was confirmed in DNA from cultured fibroblasts. Five carriers from two families had a skewed pattern (80:20-<95:5), a pattern that was found in only 11 of 148 female controls. Of the 11 carriers with a skewed pattern, the parental origin of the inactive X chromosome was maternal in all seven cases for which this could be determined. In two families, carriers with an extremely skewed pattern and carriers with a random pattern were found. The skewed X inactivation in 11 of 16 carriers is probably the result of a selection against cells with the mutated gene on the active X chromosome. Since BTHS also shows great clinical variation within families, additional factors are likely to influence the expression of the phenotype. Such factors may also influence the selection mechanism in carriers.     

Familial skewed X inactivation: a molecular trait associated with high spontaneous-abortion rate maps to Xq28.

We report a family ascertained for molecular diagnosis of muscular dystrophy in a young girl, in which preferential activation (> or = 95% of cells) of the paternal X chromosome was seen in both the proband and her mother. To determine the molecular basis for skewed X inactivation, we studied X-inactivation patterns in peripheral blood and/or oral mucosal cells from 50 members of this family and from a cohort of normal females. We found excellent concordance between X-inactivation patterns in blood and oral mucosal cell nuclei in all females. Of the 50 female pedigree members studied, 16 showed preferential use (> or = 95% cells) of the paternal X chromosome; none of 62 randomly selected females showed similarly skewed X inactivation was maternally inherited in this family. A linkage study using the molecular trait of skewed X inactivation as the scored phenotype localized this trait to Xq28 (DXS1108; maximum LOD score [Zmax] = 4.34, recombination fraction [theta] = 0). Both genotyping of additional markers and FISH of a YAC probe in Xq28 showed a deletion spanning from intron 22 of the factor VIII gene to DXS115-3. This deletion completely cosegregated with the trait (Zmax = 6.92, theta = 0). Comparison of clinical findings between affected and unaffected females in the 50-member pedigree showed a statistically significant increase in spontaneous-abortion rate in the females carrying the trait (P < .02). To our knowledge, this is the first gene-mapping study of abnormalities of X-inactivation patterns and is the first association of a specific locus for recurrent spontaneous abortion in a cytogenetically normal family. The involvement of this locus in cell lethality, cell-growth disadvantage, developmental abnormalities, or the X-inactivation process is discussed.     

Dystrophin is required for the formation of stable muscle attachments in the zebrafish embryo

A class of recessive lethal zebrafish mutations has been identified in which normal skeletal muscle differentiation is followed by a tissue-specific degeneration that is reminiscent of the human muscular dystrophies. Here, we show that one of these mutations, sapje, disrupts the zebrafish orthologue of the X-linked human Duchenne muscular dystrophy (DMD) gene. Mutations in this locus cause Duchenne or Becker muscular dystrophies in human patients and are thought to result in a dystrophic pathology through disconnecting the cytoskeleton from the extracellular matrix in skeletal muscle by reducing the level of dystrophin protein at the sarcolemma. This is thought to allow tearing of this membrane, which in turn leads to cell death. Surprisingly, we have found that the progressive muscle degeneration phenotype of sapje mutant zebrafish embryos is caused by the failure of embryonic muscle end attachments. Although a role for dystrophin in maintaining vertebrate myotendinous junctions (MTJs) has been postulated previously and MTJ structural abnormalities have been identified in the Dystrophin-deficient mdx mouse model, in vivo evidence of pathology based on muscle attachment failure has thus far been lacking. This zebrafish mutation may therefore provide a model for a novel pathological mechanism of Duchenne muscular dystrophy and other muscle diseases.     

Carrier detection of deletions in female relatives of X-linked disorders by non-isotopic in situ hybridisation.

Recent studies suggest that a non-isotopic in situ hybridisation (NISH) approach can be successfully employed to investigate the carrier status of female relatives in families of selected patients with Duchenne muscular dystrophy (DMD) or Hunter syndrome, whose diseases are due to a specific X chromosome deletion. Whilst the majority of metaphase spreads from normal females show specific hybridisation signals on both X chromosomes when tested with either dystrophin or Hunter gene-derived probes, only one X chromosome in each metaphase spread will show the relevant hybridisation complex in female carriers of deletions involving the dystrophin or Hunter gene. Thus, the NISH method can be a valuable diagnostic tool for the detection of the carrier status of female relatives of patients with X chromosome deletions.     

Duchenne muscular dystrophy (DMD) in a female with an X/autosomal translocation: Further evidence that the DMD locus is at Xp21

An isolated case of Duchenne muscular dystrophy in a female who has a de novo t(X;5)(p21;q35) translocation is described. The similarities between this patient and four previously reported females with Duchenne muscular dystrophy are discussed. It is concluded that the locus for Duchenne muscular dystrophy is at Xp21 and, furthermore, that this site may be particularly susceptible both to chromosome breakage and exchange and to gene mutation.     

Skewed X-chromosome inactivation is a common feature of X-linked mental retardation disorders

Some deleterious X-linked mutations may result in a growth disadvantage for those cells in which the mutation, when on the active X chromosome, affects cell proliferation or viability. To explore the relationship between skewed X-chromosome inactivation and X-linked mental retardation (XLMR) disorders, we used the androgen receptor X-inactivation assay to determine X-inactivation patterns in 155 female subjects from 24 families segregating 20 distinct XLMR disorders. Among XLMR carriers, ~50% demonstrate markedly skewed X inactivation (i.e., patterns 80:20), compared with only ~10% of female control subjects (P<.001). Thus, skewed X inactivation is a relatively common feature of XLMR disorders. Of the 20 distinct XLMR disorders, 4 demonstrate a strong association with skewed X inactivation, since all carriers of these mutations demonstrate X-inactivation patterns 80:20. The XLMR mutations are present on the preferentially inactive X chromosome in all 20 informative female subjects from these families, indicating that skewing is due to selection against those cells in which the XLMR mutation is on the active X chromosome.     

杜氏肌营养不良症(DMD)及其动物模型研究进展

杜氏肌营养不良症（Duchenne muscular dystrophy,DMD）属于X连锁隐性遗传病.DMD基因是人类最大基因,突变机制复杂.随着分子生物学的研究进展,对DMD的基因和其编码的抗肌萎缩蛋白（dystrophin）及抗肌萎缩蛋白相关蛋白（utrophin）的认识不断深入.本文就DMD的病理学特点,Dys基因结构、表达、功能,DMD突变及其相关检测技术,DMD实验动物模型及相关治疗的研究进展进行综述.     

Muscular dystrophy in the Japanese Spitz: an inversion disrupts the DMD and RPGR genes

An X‐linked muscular dystrophy, with deficiency of full‐length dystrophin and expression of a low molecular weight dystrophin‐related protein, has been described in Japanese Spitz dogs. The aim of this study was to identify the causative mutation and develop a specific test to identify affected cases and carrier animals. Gene expression studies in skeletal muscle of an affected animal indicated aberrant expression of the Duchenne muscular dystrophy (dystrophin) gene and an anomaly in intron 19 of the gene. Genome‐walking experiments revealed an inversion that interrupts two genes on the X chromosome, the Duchenne muscular dystrophy gene and the retinitis pigmentosa GTPase regulator gene. All clinically affected dogs and obligate carriers that were tested had the mutant chromosome, and it is concluded that the inversion is the causative mutation for X‐linked muscular dystrophy in the Japanese Spitz breed. A PCR assay that amplifies mutant and wild‐type alleles was developed and proved capable of identifying affected and carrier individuals. Unexpectedly, a 7‐year‐old male animal, which had not previously come to clinical attention, was shown to possess the mutant allele and to have a relatively mild form of the disease. This observation indicates phenotypic heterogeneity in Japanese Spitz muscular dystrophy, a feature described previously in humans and Golden Retrievers. With the availability of a simple, fast and accurate test for Japanese Spitz muscular dystrophy, detection of carrier animals and selected breeding should help eliminate the mutation from the breed.     

Co-occurrence of mutations in both dystrophin- and androgen-receptor genes is a novel cause of female Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder. Here, we report a novel mechanism for the occurrence of DMD in females. In a Vietnamese DMD girl, conventional PCR amplification analysis disclosed a deletion of exons 12–19 of the dystrophin gene on Xp21.2, with a karyotype of 46, XY. Furthermore, a novel mutation in the androgen-receptor gene on Xq11.2-q12 was identified in this girl, which led to male pseudohermaphroditism. Co-occurrence of mutations of these two genes constitutes a novel mechanism underlying female DMD.     

Mild and severe muscular dystrophy caused by a single gamma-sarcoglycan mutation.

Autosomal recessive muscular dystrophy is genetically heterogeneous. One form of this disorder, limb-girdle muscular dystrophy type 2C (LGMD 2C), is prevalent in northern Africa and has been shown to be associated with a single mutation in the gene encoding the dystrophin-associated protein gamma-sarcoglycan. The previous mutation analysis of gamma-sarcoglycan required the availability of muscle biopsies. To establish a mutation assay for genomic DNA, the intron-exon structure of the gamma-sarcoglycan gene was determined, and primers were designed to amplify each of the exons encoding gamma-sarcoglycan. We studied a group of Brazilian muscular dystrophy patients for mutations in the gamma-sarcoglycan gene. These patients were selected on the basis of autosomal inheritance and/or the presence of normal dystrophin and/or deficiency of alpha-sarcoglycan immunostaining. Four of 19 patients surveyed had a single, homozygous mutation in the gamma-sarcoglycan gene. The mutation identified in these patients, all of African-Brazilian descent, is identical to that seen in the North African population, suggesting that even patients of remote African descent may carry this mutation. The phenotype in these patients varied considerably. Of four families with an identical mutation, three have a severe Duchenne-like muscular dystrophy. However, one family has much milder symptoms, suggesting that other loci may be present that modify the severity of the clinical course resulting from gamma-sarcoglycan gene mutations.     

Expression of an X-linked muscular dystrophy in a female due to translocation involving Xp21 and non-random inactivation of the normal X chromosome

Summary A young female was diagnosed as having X-linked muscular dystrophy of the Duchenne type. Chromosome studies, including trypsin-Giemsa banding, Quinacrine fluorescence, and nucleolus organizer region (NOR) silver staining revealed an X-autosome reciprocal translocation t(X;21) (p21;p12). Utilizing both [³H] thymidine autoradiography and the BrdU-Hoechst 33258-Giemsa technique, lymphocytes and fibroblasts were found to show a preferential inactivation of the normal X suggesting the presence of a single mutant gene on the translocated X. This patient is one of seven reported cases of an X-linked muscular dystrophy associated with an X-autosome translocation. In all seven cases the exchange point in the X chromosome is in band p21 at or near the site of the Duchenne gene.     

女性Duchenne/Becker型肌营养不良症携带者发病机理的研究进展

Duchenne/Becker型肌营养不良(DMD/BMD)是一类常见的X连锁隐性遗传病,多见于男性患者,女性携带者一般不发病,因为女性体内会发生随机的X染色体失活,而使体内呈现镶嵌型。目前,越来越多的文献报道DMD/BMD女性携带者发病的病例,其症状有轻有重,但发病机制尚不明了,大多数研究认为与X染色体的偏斜失活有关,即携带DMD突变的X染色体异常活化,使正常DMD基因弱或无表达,从而无法生成正常功能的dystrophin蛋白,表现为DMD/BMD。本文主要综述了X偏斜失活与DMD女性携带者发病相关性的研究进展。