In utero fetal muscle biopsy for the diagnosis of Duchenne muscular dystrophy

Deoxyribonucleic acid techniques can be used to diagnose Duchenne muscular dystrophy prenatally in male fetuses that are at risk. Deoxyribonucleic acid-based prenatal diagnosis can be impossible when there is only one prior affected male and there is no identifiable deletion or alteration. We performed fetal muscle biopsy in utero in such a case and documented the presence of dystrophin, thereby confirming normality in a male fetus at risk. This first in utero experience adds fetal muscle biopsy to the available procedures for fetal tissue diagnosis.     

Fetal muscle biopsy as a diagnostic tool in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a relentless progressive disorder, leading to severe disability during childhood and death in adolescence or early adulthood. In most families, prenatal diagnosis is readily achieved by molecular detection of DNA deletions using chorionic villi or amniocytes, or by linkage analysis. In some cases, however, molecular methods fail to provide a definitive diagnosis and in such cases in utero fetal muscle biopsy may serve as a diagnostic option. We describe three families in whom fetal muscle biopsy was performed, focusing on the prenatal diagnostic dilemmas, the indications and timing for in utero fetal muscle biopsy, and the difficulties encountered.     

Maternal contamination at fetal muscle biopsy

Duchenne muscular dystrophy (DMD) can be diagnosed by fetal muscle biopsy and immunohistochemical staining showing the absence of dystrophin. We report a case of fetal muscle biopsy in which the needle gun was successfully fired within the fetal gluteal muscle but the sample was contaminated by maternal tissue. This was attributed to the design of the biopsy needle, allowing transient opening of the biopsy core as the needle penetrated the maternal rectus sheath, muscle, and myometrium. Histology showed tissue suggestive of maternal origin, confirmed by DNA analysis. Repeat sampling revealed fetal muscle with normal dystrophin expression. We recommend that care be taken during needle insertion to avoid maternal contamination, and tests be used to confirm the fetal source of the sample.     

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。     

Triplet pregnancy with acardius acranius after preimplantation diagnosis

Objective: To report the first case of fetal malformation after preimplantation diagnosis for Duchenne muscular dystrophy (DMD).

Design: Case report.

Setting: Perinatal center in a university hospital.

Patient(s): A conductor for DMD in her third pregnancy.

Intervention(s): Preimplantation diagnosis was performed in an outside hospital. In our center, a dichorionic triplet pregnancy with acardius acranius was diagnosed. The anastomosis between the “pump”-twin and the fetus with acardius was embolized with histoacryl to prevent worsening cardiac insufficiency of the “pump”-twin.

Main Outcome Measure(s): Pregnancy outcome.

Result(s): The anastomosis between the “pump”-twin and the fetus with acardius was occluded successfully. Premature preterm rupture of membranes led to rapid labor and delivery at 24 + 5 weeks’ gestation. The smaller girl died of severe hyaline membrane disease, whereas the other infant had no major clinical problems and has developed well.

Conclusion(s): There might be an association between embryo biopsy and fetal malformations. The setting up of a birth register after embryo biopsy is strongly recommended.     

Prenatal Diagnosis of Fetal Disorders Part I: Technological Capabilities

:The indications, techniques, and results of amniocentesis, sonography, fetos-copy, and maternal serum screening for alpha-fetoprotein are described, along with some of the controversies over interpretation of studies. Down syndrome, neural tube defects, sickle cell anemia, hemophilia, Duchenne muscular dystrophy, alpha-thalassemia, beta-thalassemia, galactosemia, and Toy Sachs disease are among the genetic disorders for which such diagnosis is sought. The diagnosis of cystic fibrosis and studies of the efficacy and safety of first trimester diagnosis by chorionic biopsy are two directions of future promise. (BIRTH 10:4, Winter 1983)     

Dystrophin protein and RFLP analysis for fetal diagnosis and carrier confirmation of Duchenne muscular dystrophy

A pregnant woman with indeterminate Duchenne muscular dystrophy (DMD) carrier status, but with DMD diagnosed in her deceased brother (unavailable for study), presented for prenatal diagnosis, intending to continue the pregnancy only if proven unaffected with DMD with near absolute certainty. Creatine kinase (CK) assays to clarify carrier status were inconclusive. Male sex in the fetus was identified, but DNA restriction fragment length polymorphism (RFLP) analysis was not yet available to this centre to investigate the possible transmission of the DMD gene, and the pregnancy was terminated. Tissue histology and dystrophin protein analysis demonstrated the absence of DMD. In a situation with proven maternal carrier status, future fetal inheritance of the opposite maternal X chromosome would indicate the presence of DMD. However, maternal carrier status remained in doubt through a second pregnancy, even with RFLP studies, and was finally established when dystrophin analysis confirmed the presence of DMD in the second fetus. Histologic findings are presented, contrasting features in the two fetuses. The value of dystrophin analysis for establishing the diagnosis of fetal DMD, in this case proving maternal carrier status in a difficult situation, and for demonstrating DMD gene:RFLP haplotype relationships is illustrated.     

Prenatal diagnosis in a female carrying a deletion close to the Duchenne locus

Family studies including the proband are usually needed before a prenatal diagnosis may be performed for Duchenne muscular dystrophy. We report here on prenatal diagnosis in a family where the solitary index case was dead, and where the consultand and her mother were assumed to be carriers by independent evidence. DNA analysis revealed that both the consultand and her mother had an X chromosome deleted for DNA material in the Xp21 region. The female fetus also carried the deleted X chromosome.     

Complex genetic counseling and exclusion of duchenne muscular dystrophy in a twin pregnancy after in vitro fertilization (IVF)

A twin pregnancy following in vitro ferilization-embryo transfer coincidentally at risk for the X-linked recessive Duchenne muscular dystrophy is described. Firsttrimester prenatal diagnosis by transabdominal chorionic villus samplings on the dichorionic placentae and molecular linkage analysis could exclude the disorder in both fetuses. Genetic counseling and prenatal diagnosis were particularly complex due to the twin pregnancy, the need for linkage analysis, and confined placental mosaicism 45,X/46XX in one of the fetuses. All parties should be aware that additional invasive diagnostic procedures in the second trimester might be required. It is proposed that, in similar situations, only one, arguably two, fertilized egg be transferred at a time to facilitate prenatal diagnosis and decision making for these rare couples. This problem, however, may be increasingly overcome by preimplantation diagnosis.     

Dystrophin immunostaining of muscle from Chinese patients with various neuromuscular diseases.

The localization of dystrophin was studied using the immunohistochemical method of diagnostic muscle specimens from 68 patients, aged 9 days to 65 years, with various neuromuscular disorders. Additionally muscle specimens from 2 normal humans and 2 normal mice were used as positive controls, and those from 2 mice with x-linked muscular dystrophy as negative controls. The specimens from all 14 Duchenne muscular dystrophy (DMD) patients, including one with preclinical DMD, showed negative dystrophin staining except for two which had 0.2% to 0.8% positive fibers. The mdx mice also showed negative dystrophin staining. In Becker muscular dystrophy (BMD), muscle fibers stained in a patchy or discontinuous fashion. Two symptomatic DMD carriers exhibited a distinct mosaic pattern of dystrophin positive and negative fibers. In contrast, dystrophin was present in all 7 biopsies from patients with 4 other types of muscular dystrophy (limb-girdle, congenital, myotonic and facioscapulohumeral). Other specimens, those from normal humans and control mice, revealed homogeneous immunostaining along the surface membranes of all muscle fibers. We thus conclude that immunohistochemical dystrophin staining can aid in differentiating DMD from preclinical DMD or BMD, as well as in the detection of DMD carriers.     

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。     

Pitfalls in prenatal diagnosis of DMD due to placental mosaicism of the X-chromosomes: prenatal and postnatal findings in a fetus with a deletion of exons 67-71 of the dystrophin gene

Prenatal diagnosis of Duchenne and Becker muscular dystrophy (DMD) is performed as a routine procedure in many laboratories. The major potential problem is an incorrect diagnosis that could be obtained due to contamination with maternal tissue. We report a case of mosaicism of the X-chromosomes confined to the placenta as a possible source of confusing results in prenatal diagnosis of DMD. To the best of our knowledge, this is the first reported case of this problem in a prenatal DMD diagnosis.     

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.     

False elevation of amniotic fluid enzymes of relevance for prenatal diagnosis: creatine kinase measurement in relation to Duchenne muscular dystrophy

The creatine kinase activity of amniotic fluid was measured in samples collected at fetoscopy. In our first study, the control sample range was 0.25 IU/l, although four samples had activities of 35-85 IU/l. Elevated values did not correlate with the activities in the fetal or maternal circulations. Electrophoresis revealed the presence of the BB isozyme of creatine kinase rather than just the MM form as expected. This suggested that the source of the elevated enzyme activity was from the myometrium, damaged by insertion of the trocar and cannula. In a further series the first 2 ml of amniotic fluid withdrawn yielded a much higher creatine kinase activity than a second aliquot. A control series of such second samples (first 2 ml discarded) gave an activity range of 0-7 IU/l with no spuriously high values. This compares favourably with a series from single samplings taken by amniocentesis. Normal creatine kinase activities were found in the amniotic fluids from 20 pregnancies at risk of Duchenne muscular dystrophy. We conclude that for accurate measurement of amniotic fluid enzyme activity the first portion withdrawn should be discarded. Amniotic fluid creatine kinase activity is of no value for the prenatal diagnosis of Duchenne muscular dystrophy.     

Prenatal diagnosis using deletion studies in Duchenne muscular dystrophy

Accurate carrier testing and prenatal diagnosis in Duchenne muscular dystrophy (DMD) families is facilitated when an Xp21 deletion is found to be segregating within a family. We discuss the results of the DNA testing in two families, one in which DNA from affected males was available for study and the other in which no DNA from an affected male was available. Factors complicating the counselling of DMD deletion families are outlined.     

gamma-sarcoglycan deficiency muscular dystrophy in two adults.

All dystrophin-associated proteins contain sarcoglycan complex. Different forms of muscular dystrophy are caused by defective expression of different proteins of this structure. gamma-Sarcoglycan deficiency muscular dystrophy, so-called severe childhood autosomal recessive muscular dystrophy (SCARMD), is a rare disease that has not been previously reported in Taiwan. This paper describes two Taiwanese adults with this disease: a 26-year-old man with calf pseudohypertrophy who had weakness in both legs for 1 year; and a 43-year-old woman who had progressive weakness in all four limbs, with the initial symptom of gait disturbance at the age of 32 years. Analysis of muscle biopsy specimens, which showed total deficiency of gamma-sarcoglycan protein on immunostaining, confirmed the diagnosis of SCARMD in both cases. However, the clinical manifestations in these two patients, including lower proximal limb weakness initially developing in adulthood with a slow progressive course, are different from previously reported cases of SCARMD. The literature on this disease is reviewed and possible mechanisms of these distinct clinical presentations are discussed.     

Rapid prenatal diagnosis of Duchenne muscular dystrophy with gene duplications by ion-pair reversed-phase high-performance liquid chromatography coupled with competitive multiplex polymerase chain reaction strategy

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by various mutations in the dystrophin gene. Rapid prenatal diagnosis of DMD with gene duplications is difficult due to limitation in gene dosage determination and the requirement for a known disease-causing mutation in the pedigree to achieve a rapid and accurate diagnosis. We report, here, a case with rapid prenatal diagnosis of DMD-affected male with gene duplications in the absence of a known disease-causing variation in the pedigree by using ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) coupled with competitive multiplex polymerase chain reaction (PCR) protocol. In cases with clinical diagnosis of DMD/BMD, this test should identify greater than 92% of disease-causing DNA variants. The postnatal genetic diagnosis of this case and the same disease-causing mutations subsequently identified in other members of the pedigree confirmed the accuracy of competitive multiplex PCR/IP-RP-HPLC assay in direct prenatal diagnosis of DMD.     

Gene therapy for the fetus: is there a future?

Gene therapy uses the intracellular delivery of genetic material for the treatment of disease. A wide range of diseases - including cancer, vascular and neurodegenerative disorders and inherited genetic diseases - are being considered as targets for this therapy in adults. There are particular reasons why fetal application might prove better than application in the adult for treatment, or even prevention of early-onset genetic disorders such as cystic fibrosis and Duchenne muscular dystrophy. Research shows that gene transfer to the developing fetus targets rapidly expanding populations of stem cells, which are inaccessible after birth, and indicates that the use of integrating vector systems results in permanent gene transfer. In animal models of congenital disease such as haemophilia, studies show that the functionally immature fetal immune system does not respond to the product of the introduced gene, and therefore immune tolerance can be induced. This means that treatment could be repeated after birth, if that was necessary to continue to correct the disease. For clinicians and parents, fetal gene therapy would give a third choice following prenatal diagnosis of inherited disease, where termination of pregnancy or acceptance of an affected child are currently the only options. Application of this therapy in the fetus must be safe, reliable and cost-effective. Recent developments in the understanding of genetic disease, vector design, and minimally invasive delivery techniques have brought fetal gene therapy closer to clinical practice. However more research needs to be done in before it can be introduced as a therapy.     

Incidental prenatal detection of an Xp deletion using an anonymous primer pair for fetal sexing

We report on the incidental prenatal detection of an interstitial X-chromosomal deletion in a male fetus and his mother by fetal sexing with a primer pair recognizing an X-Y homologous locus (DXYS19), formerly unassigned on the X chromosome. The proband asked for prenatal diagnosis because of her elevated age and risk of Duchenne muscular dystrophy (DMD). Prior to molecular genetic testing for DMD, fetal sexing was carried out on DNA prepared from cultured amniocytes. PCR analysis revealed the expected Y-chromosomal product, but did not show the constitutive X-chromosomal fragment. The absence of the X-chromosomal fragment in the fetus and on one X chromosome of the mother was confirmed by Southern hybridization of HindIII restricted DNA with probe pJA1165 (DXYS19). DXYS19X was mapped to Xp22.3 by combining several approaches, including: (1) analysis of somatic cell hybrid lines containing different fragments of the human X chromosome; (2) Southern hybridization of a yeast artificial chromosome (YAC)-filter panel provided by the Resource Center/Primary Database (RZPD); (3) FISH analysis; and (4) re-evaluation of two patients with interstitial deletions in Xp22.3. The extent of the deletion in the fetus was estimated by further markers from Xp22.3 and found to include the STS gene. Mental retardation could not be excluded since some mentally retarded patients exhibit overlapping deletions.     

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.