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。     

Proteomics profiling of urine reveals specific titin fragments as biomarkers of Duchenne muscular dystrophy

Diagnosis of muscular dystrophies is currently based on invasive methods requiring muscle biopsies or blood tests. The aim of the present study was to identify urinary biomarkers as a diagnostic tool for muscular dystrophies. Here, the urinary proteomes of Duchenne muscular dystrophy (DMD) patients and healthy donors were compared with a bottom-up proteomic approach. Label-free analysis of more than 1100 identified proteins revealed that 32 of them were differentially expressed between healthy controls and DMD patients. Among these 32 proteins, titin showed the highest fold change between healthy subjects and DMD patients. Interestingly, most of the sequenced peptides belong to the N-terminal and C-terminal parts of titin, and the presence of the corresponding fragments in the urine of DMD patients was confirmed by Western blot analysis. Analysis of a large cohort of DMD patients and age-matched controls (a total of 104 individuals aged from 3 to 20 years) confirmed presence of the N-ter fragment in all but two patients. In two DMD patients aged 16 and 20 years this fragment was undetectable and two healthy controls of 16 and 19 years with serum CK >800 IU/L demonstrated a low level of the fragment. N- and C-terminal titin fragments were also detected in urine from patients with other muscular dystrophies such as Becker muscular dystrophy and Limb-girdle muscular dystrophy (type 1D, 2D and 2J) but not in neurogenic spinal muscular atrophy. They were also present in urine of dystrophin-deficient animal models (GRMD dogs and mdx mice). Titin is the first urinary biomarker that offers the possibility to develop a simple, non-invasive and easy-to-use test for pre-screening of muscular dystrophies, and may also prove to be useful for the non-invasive follow up of DMD patients under treatment.     

Rapid in vivo measurement of the topography of human skin by active image triangulation using a digital micromirror device

Background/aims: Topometry is one of the most relevant methods for biophysical research on skin in dermatologic and cosmetic science, because it relates very closely to the perceived quality of skin. Taking silicon replicas of skin sites under investigation and measuring those imprints with mechanical or optical profilometers is still the most frequently used method. Direct measurement of the topography of human skin in vivo by active image triangulation avoids the need to make replicas and seems to be a promising alternative. Methods: The introduction of active image triangulation in conjunction with phase-shift techniques in skin topometry enables a fast and non-invasive measurement of the skin surface in vivo. The main attribute of the proposed system is the projection of a regular sinusoidal intensity pattern with a sophisticated digital projection device onto the surface of skin under a certain angle of incidence. The height information of the structured surface is coded in the distorted intensity pattern, which is recorded by an appropriate video technique. Results: Successful in vivo measurements of selected body sites and measurements on scar, nevus, wound and wrinkles are presented in this paper. Furthermore, irritation of skin, influence of hydration of skin, and differences between youthful and elderly skin can be detected in the measurement results of the new optical system. Conclusions: For measuring the topog raphy of human skin, active image triangulation is appropriate both for macrotopometry (nevus, scar, wound) and for microtopometry (casts, selected body sites). This new non-contact technique allows dynamic measurements of alterations in skin topography as a consequence of certain treatments (e.g., application of ingredient, hydration of skin) without removal of corneocytes or scales. Optical three-dimensional (3D) topometry using active image triangulation appears to offer a significant improvement in speed and flexibility, providing a fast and accurate analysis of skin surface topography.     

Duchenne muscular dystrophy: Recent concepts

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease, affecting only males and transmitted by females. Recent proposed pathogeneses are defective DNA repair mechanism and cell-mediated cytotoxicity. Serum CPK estimation is most frequently high (30–300) and is also reliable for detecting carriers. It is important to recognise the affected boys as early as possible. Diagnosis of DMD is made from clinical features, CPK estimation, EMG and ECG abnormalities. The use of agents to reduce influx of intracellular calcium in patients with DMD are emerging for therapeutic consideration. New advance in genetic counselling is the localisation of the gene for DMD using DNA polymorphisms.     

Prevalence and psychosocial impact of lower urinary tract symptoms in patients with Duchenne muscular dystrophy

Patients with Duchenne muscular dystrophy (DMD) frequently report lower urinary tract symptoms at the outpatient rehabilitation clinic. The purpose of this study was to determine the prevalence of lower urinary tract symptoms in the Dutch male DMD population and their effect on quality of life. A postal questionnaire was sent to members of Dutch DMD patient organisations. 199 male patients with confirmed DMD and over the age of 3 years were included. 170/199 (85%) patients reported one or more lower urinary tract symptoms. Generally, post micturition dribble, straining and feeling of incomplete emptying were most frequently mentioned. 42% of patients (range 18–76%) experienced the symptoms as a problem. In 49/170 (29%) patients, it reduced quality of life. In conclusion, lower urinary tract symptoms in DMD patients are under reported and under diagnosed. However, the vast majority of male DMD patients with symptoms experience them as a problem, often reducing quality of life.     

X-irradiation improves mdx mouse muscle as a model of myofiber loss in DMD

The mdx mouse, although a genetic and biochemical homologue of human Duchenne muscular dystrophy (DMD), presents a comparatively mild histopathological and clinical phenotype. These differences are partially attributable to the greater efficacy of regeneration in the mdx mouse than in DMD muscle. To lessen this disparity, we have used a single dose of X-irradiation (16 Gy) to inhibit regeneration in one leg of mdx mice. The result is an almost complete block of muscle fiber regeneration leading to progressive loss of muscle fibers and their replacement by loose connective tissue. Surviving fibers are mainly peripherally nucleated and, surprisingly, of large diameter. Thus, X-irradiation converts mdx muscle to a model system in which the degenerative process can be studied in isolation from the complicating effect of myofiber regeneration. This system should be of use for testing methods of alleviating the myofiber degeneration which is common to mdx and DMD.     

Diagnostic utility of skin biopsy in dystrophinopathies

Aims

Muscle biopsy is an important diagnostic modality and screening test for the diagnosis of dystrophinopathies. Sometimes muscle biopsies are needed for the diagnosis when genetic tests are inconclusive and are also useful for immunoblotting assay of the dystrophin protein. However, the procedure is painful, requires anesthesia and sometimes needs to be repeated. This study was undertaken to elucidate the role of skin biopsy in the diagnosis of dystrophinopathies and to validate if it can be utilized as a useful adjunct/replacement for the muscle biopsy.

Methods

Paired skin and muscle biopsies were studied from 39 patients with Duchenne muscular dystrophy (DMD), 4 patients with Becker's muscular dystrophy (BMD) and 37 controls. Immunostaining for dystrophin and utrophin was done on frozen sections of the test group and controls and their staining pattern in skin biopsies was compared with corresponding muscle biopsies.

Results

Immunostaining for dystrophin was negative in the skin biopsies of all patients (39/39, 100%) with DMD and was only weakly expressed in skin of BMD patients (4/4, 100%). Dystrophin was strongly expressed on arrector pili muscle cells of all control patients (94.6%) except two cases in whom it was weakly expressed. Utrophin was expressed on the arrector pili muscle cells of DMD patients (39/39, 100%) as well as controls (30/37, 81.1%).

Conclusion

Our study suggests that skin biopsy is very useful for the diagnosis of dystrophinopathies and their differentiation from other muscle diseases. It has high degrees of sensitivity, specificity, and positive and negative predictive values. It can be a useful adjunct/replacement for the muscle biopsy especially when repeated biopsies are required for monitoring therapy or in patients with advanced DMD where extreme fibrosis, adipose tissue infiltration and inflammation make interpretation of the muscle biopsy difficult. Skin biopsy is a simple, cost effective, less invasive and less traumatic diagnostic procedure when compared with muscle biopsy. This is even more pertinent because patients with muscular dystrophies have a higher risk for any form of general anesthesia. A smaller scar and fewer chances of infection at the site of biopsy are other additional advantages of skin biopsy over muscle biopsy.     

DMD exon 1 truncating point mutations: Amelioration of phenotype by alternative translation initiation in exon 6

Mutations in the DMD gene result in two common phenotypes associated with progressive muscle weakness: the more severe Duchenne muscular dystrophy (DMD) and the milder Becker muscular dystrophy (BMD). We have previously identified a nonsense mutation (c.9G>A; p.Trp3X) within the first exon of the DMD gene, encoding the unique N‐terminus of the 427‐kDa muscle isoform of the dystrophin protein. Although this mutation would be expected to result in severe disease, the clinical phenotype is very mild BMD, with ambulation preserved into the seventh decade. We identify the molecular mechanism responsible for the amelioration of disease severity to be initiation of translation at two proximate AUG codons within exon 6. Analysis of large mutational data sets suggests that this may be a general mechanism of phenotypic rescue for point mutations within at least the first two exons of the DMD gene. Our results directly demonstrate, for the first time, the use of alternate translational initiation codons within the DMD gene, and suggest that dystrophin protein lacking amino acids encoded by the first five exons retains significant function. Hum Mutat 0:1–8, 2009. © 2009 Wiley‐Liss, Inc.     

Mutational spectrum of DMD mutations in dystrophinopathy patients: application of modern diagnostic techniques to a large cohort

Mutations in the DMD gene, encoding the dystrophin protein, are responsible for the dystrophinopathies Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD), and X-linked Dilated Cardiomyopathy (XLDC). Mutation analysis has traditionally been challenging, due to the large gene size (79 exons over 2.2 Mb of genomic DNA). We report a very large aggregate data set comprised of DMD mutations detected in samples from patients enrolled in the United Dystrophinopathy Project, a multicenter research consortium, and in referral samples submitted for mutation analysis with a diagnosis of dystrophinopathy. We report 1,111 mutations in the DMD gene, including 891 mutations with associated phenotypes. These results encompass 506 point mutations (including 294 nonsense mutations) and significantly expand the number of mutations associated with the dystrophinopathies, highlighting the utility of modern diagnostic techniques. Our data supports the uniform hypermutability of CGA>TGA mutations, establishes the frequency of polymorphic muscle (Dp427m) protein isoforms and reveals unique genomic haplotypes associated with “private” mutations. We note that 60% of these patients would be predicted to benefit from skipping of a single DMD exon using antisense oligonucleotide therapy, and 62% would be predicted to benefit from an inclusive multiexonskipping approach directed toward exons 45 through 55. Hum Mutat 30:1657–1666, 2009. © 2009 Wiley-Liss, Inc.