Dystrophin protein and rflp analyis 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.     

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. Copyright © 2007 John Wiley & Sons, Ltd.     

Normal pregnancy after preimplantation DNA diagnosis of a dystrophin gene deletion

To perform preimplantation DNA diagnosis for Duchenne muscular dystrophy (DMD) in a female carrier of a dystrophin gene deletion of exons 3–18, we developed a polymerase chain reaction (PCR)-based assay of exon 17 sequences. Exon 17 was efficiently amplified in all 50 single blastomeres of normal control embryos and in five blastomeres of one male embryo of the DMD carrier obtained after a first preimplantation diagnosis (PID) for gender determination. In ten blastomeres of another two male embryos of the DMD carrier, no PCR signals were observed, probably as a result of the deletion. After intracytoplasmic sperm injection, embryos were analysed for exon 17 and three of the four embryos showing normal PCR signals were replaced, resulting in a singleton pregnancy. Prenatal diagnosis showed a female karyotype and DNA analysis indicated that the fetus was not a DMD carrier.     

Dystrophin gene analysis and prenatal diagnosis of Duchenne muscular dystrophy in Russia

Of 126 families referred for counselling of Duchenne muscular dystrophy (DMD), DNA analysis has been suggested to 119 families with at least one affected child or with an affected close male relative of the woman at risk of being a DMD carrier. A large proportion (about 80 per cent) of the families were represented by sporadic cases (only one affected individual). By means of multiplex polymerase chain reactions with different sets of oligoprimers providing amplification of 10–11 different exons, altogether 49 dystrophin gene deletions were identified (41 per cent). Eighteen deletions clustered in the 5′ ‘hot spot’ region of DMD cDNA and 36 in the distal half of the central rod domain around exons 43–53. An unusually high frequency (18 per cent) of deletions involving exons 17–19 was discovered. Large deletions extending through both ‘hot spot’ regions and thus occupying over 30–40 exons were recorded in five cases (10 per cent). Seventy-six of 94 families were found to be informative by RFLP analysis for intragenic or extragenetic DNA probes. Carrier status was ascertained in 20 and rejected in 32 female relatives in 40 DMD families. Eight DMD-affected fetuses were diagnosed prenatally by direct deletion testing or by RFLP analysis. Feasible interpopulation variations in the dystrophin gene deletion pattern are discussed. The prospects for more effective prenatal diagnosis and carrier detection in high-risk DMD families in Russia are briefly outlined.     

Carrier detection and microsatellite analysis of duchenne and becker muscular dystrophy in spanish families

Duchenne and Becker muscular dystrophy (D/BMD) are usually problematical when trying to determine the carrier status of at-risk women, which usually has to be based on haplotype or dosage analysis on Southern blots. Using multiplex polymerase chain reaction (PCR) analysis, we have detected deletions in 20 out of 44 D/BMD families with living affected members (45·5 per cent), more often in sporadic cases of DMD (14/22 with detectable deletion) than in familial ones (4/15), the majority (15/20) occurring in the distal region of the D/BMD gene. Four highly informative short tandem repeat polymorphisms (STRPs), which lie within the distal deletion hot spot of the D/BMD gene, can show loss of heterozygosity in carrier females, providing direct evidence of their carrier status. These STRPs greatly improve informativity, with a combined heterozygosity of 100 per cent and with the majority of families informative for three of the four STRPs. In 14/15 (93 per cent) of the families with distal deletions, the STRPs provided direct information on carrier status, and in some cases, they provide valuable information on recombination breakpoints and non-paternity.     

Identification of duchenne muscular dystrophy genomic probe P20 constant Taq1 fragment corresponding to the EcoRV and Msp1 polymorphisms

The majority of Duchenne and Becker muscular dystrophy cases are caused by deletions observable in Southern blots with cDNA probes for the gene. When the deletion includes polymorphic probes, they may be used to determine carrier status by deletion segregation analysis: non-inheritance of parental alleles, or heterozygosity. The polymorphic genomic probe P20 is deleted in a large percentage of probands. P20 hybridizes with two constant fragments of 6.7 and 0.8 kb in Taql digests. In a number of probands, only the larger P20 Taq1 fragment is deleted. This study demonstrates that this fragment corresponds with the polymorphic EcoRV and Mspl fragments of P20. Families in which the upper Taql fragment is deleted may be screened for carrier status using non-inheritance of parental alleles or heterozygosity of P20 in EcoRV or Mspl digests.     

Carrier detection of duchenne muscular dystrophy through analysis of dna from deciduous teeth of a dead affected child

The sister of a child affected by Duchenne muscular dystrophy (DMD) was referred for genetic counselling to assess the risk of her being a carrier. Her brother had died 15 years previously at the age of 8. There were no other affected males in the family. There were no methods for DNA investigation at the time of the child's death and the family had never been studied for linkage with polymorphic probes on the chromosomal region Xp21. The only tissue from which an assessment of the risk could be made by DNA linkage analysis was two of the child's deciduous teeth that the parents had kept. DNA was extracted using a protocol described for the recovery of ancient DNA from museum specimens and archaeological finds. Multiplex amplification did not reveal deletions in 19 exons spanning the hot-spot regions for deletions within the dystrophin gene in Xp21. Linkage analysis using three highly polymorphic microsatellites demonstrated that the sister had not received the X chromosome borne by her brother. These results show that DNA extracted from teeth is a reliable source for molecular diagnosis.     

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/1, although four samples had activities of 35–85 IU/1. 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/1 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 for 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.     

Prenatal exclusion of duchenne muscular dystrophy by fetal muscle biopsy

A fetal thigh muscle biopsy was performed at 18 weeks and 6 days' gestation using an automatic 18-gauge biopsy needle. A positive immunoreaction with antisera to the amino- and carboxy-terminals of dystrophin excluded Duchenne muscular dystrophy from this at-risk male.     

Lesch—Nyhan syndrome: Carrier and prenatal diagnosis

We report the results of carrier and prenatal diagnosis for hypoxanthine guanine phosphoribosyltransferase (HPRT) deficiency, Lesch—Nyhan syndrome, by carrier testing of 83 women and prenatal analysis of 26 pregnancies. Our diagnostic methodologies include mutation detection and linkage analysis for probands and their families and biochemical measurement of HPRT enzyme activity for at-risk pregnancies. Identification of the mutation in the index case of each family permits precise carrier diagnosis using polymerase chain reaction (PCR) amplification of HPRT gene sequences and automated DNA sequencing. We demonstrate 100 per cent sensitivity for the detection of mutations in the HPRT gene of affected males and highly efficient carrier testing of at-risk females. Two other molecular methods proven to have high utility include PCR-based dosage analysis and linkage analysis by PCR amplification of a short tandem repeat (STR) in intron 3 of the HPRT gene. As a result, 45 at-risk women, 56 per cent of those tested, were identified not to be carriers of their family's HPRT gene mutation. Seven of these women were the mothers of affected males and prenatal testing for future pregnancies was recommended because of the possibility of gonadal mosaicism. Thirty-eight of these women were more distant relatives of affected males, thereby eliminating the need for future prenatal procedures. These studies illustrate the utility and precision of molecular methodologies for carrier and prenatal diagnosis of Lesch—Nyhan syndrome. These studies also illustrate that molecular diagnostic studies of affected males and carrier testing prior to pregnancy can clarify genetic risk predictions and eliminate unnecessary prenatal procedures.     

Case report: birth after preimplantation genetic diagnosis of a subtle mutation in SMN1 gene

Spinal muscular atrophy (SMA) preimplantation genetic diagnosis (PGD) has been available since 1998. Protocols are based on the detection of the homozygous deletion of exon 7, which are present in 90–98% of SMA patients. A couple where the woman was a heterozygous carrier of the usual SMN1 Del7 mutation and the man was a heterozygous carrier of pMet263Arg substitution in exon 6 of SMN1 gene was referred for PGD. The usual PGD test being unsuitable for this couple, we developed a novel duplex polymerase chain reaction (PCR)-based PGD test for the detection of the mutation pMet263Arg by allele specific amplification, combined with the amplification of D5S641 extragenic polymorphic marker. PCR conditions were established using single control lymphoblasts and lymphocytes from the pMet263Arg substitution carrier. Amplification was obtained in 100% of the 86 single cells tested, amplification refractory mutation system (ARMS) PCR was specific in 100% of single cells tested and a complete genotype (mutation plus D5S641) was achieved in 88% of them. A PGD cycle was performed successfully and a pregnancy was obtained. An unaffected girl was born and postnatal diagnosis confirmed PGD results. This is the first PGD described for SMA because of another mutation than the major homozygous exon 7 deletion of SMN1. In the future, a similar strategy could be adopted for other subtle mutations of this gene. Copyright © 2006 John Wiley & Sons, Ltd.     

Molecular prenatal diagnosis: the impact of modern technologies

Originally prenatal diagnosis was confined to the diagnosis of metabolic disorders and depended on assaying enzyme levels in amniotic fluid. With the development of recombinant DNA technology, molecular diagnosis became possible for some genetic conditions late in the 1970s. Here we briefly review the history of molecular prenatal diagnostic testing, using Duchenne muscular dystrophy as an example, and describe how over the last 30 years we have moved from offering testing to a few affected individuals using techniques, such as Southern blotting to identify deletions, to more rapid and accurate PCR-based testing which identifies the precise change in dystrophin for a greater number of families. We discuss the potential for safer, earlier prenatal genetic diagnosis using cell free fetal DNA in maternal blood before concluding by speculating on how more recent techniques, such as next generation sequencing, might further impact on the potential for molecular prenatal testing. Progress is not without its challenges, and as cytogenetics and molecular genetics begin to unite into one, we foresee the main challenge will not be in identifying the genetic change, but rather in interpreting its significance, particularly in the prenatal setting where we frequently have no phenotype on which to base interpretation. Copyright © 2010 John Wiley & Sons, Ltd.     

Creatine kinase estimation in pure fetal blood samples for the prenatal diagnosis of duchenne muscular dystrophy

This paper compares the results of a survey of plasma creatine kinase (CK) activity measured in fetuses at-risk for Duchenne muscular dystrophy (DMD) with a reliable control series. Only pure fetal blood samples obtained by fetoscopy at between 17–24 weeks gestational age were used. Of the at-risk group 19 male pregnancies, mostly at low risk for DMD, proceeded to term with a normal outcome; there was no significant difference between their fetal plasma CK activities and the control group. Another 21 male pregnancies were terminated. This group included the highest risk mothers and hence was expected to contain a significant proportion of affected fetuses. The fetal plasma CK activity range was overlapping but significantly higher than the control group. No grossly elevated CK value was obtained. We conclude that, on average, DMD fetuses at this gestational age have higher plasma CK activity than controls. The problems of applying this finding to the prenatal diagnosis of DMD are discussed.     

Community attitudes to cystic fibrosis carrier testing in England: A pilot study

Inexpensive and accurate carrier testing for cystic fibrosis (CF) will be possible in the near future. There are no existing studies on the attitudes of English persons in the community to carrier testing for CF or any other recessive disorder. We have conducted a trial study of 166 persons at two schools, two doctors' surgeries, and a family planning clinic. Only a minority had clear pre-existing knowledge of cystic fibrosis and its genetic nature. However, over 80 per cent of those questioned expressed interest in knowing their carrier status. Although it is well known that uptake can only be assessed when a service is in place, and while further studies are required to confirm that testing will be of interest to couples of reproductive age with no previous knowledge of CF, the data strongly suggest that there will be interest in communitywide testing for CF carrier status when such a test becomes available in the United Kingdom.     

Distal mdx muscle groups exhibiting up-regulation of utrophin and rescue of dystrophin-associated glycoproteins exemplify a protected phenotype in muscular dystrophy

Unique unaffected skeletal muscle fibres, unlike necrotic torso and limb muscles, may pave the way for a more detailed understanding of the molecular pathogenesis of inherited neuromuscular disorders and help to develop new treatment strategies for muscular dystrophies. The sparing of extraocular muscle in Duchenne muscular dystrophy is mostly attributed to the special protective properties of extremely fast-twitching small-diameter fibres, but here we show that distal muscles also represent a particular phenotype that is more resistant to necrosis. Immunoblot analysis of membranes isolated from the well established dystrophic animal model mdx shows that, in contrast to dystrophic limb muscles, the toe musculature exhibits an up-regulation of the autosomal dystrophin homologue utrophin and a concomitant rescue of dystrophin-associated glycoproteins. Thus distal mdx muscle groups provide a cellular system that naturally avoids myofibre degeneration which might be useful in the search for naturally occurring compensatory mechanisms in inherited skeletal muscle diseases.     

Rapid molecular prenatal diagnosis of ataxia-telangiectasia by direct mutational analysis

Mutations of the ataxia-telangiectasia-mutated (ATM) gene are responsible for the autosomal recessive disorder ataxia-telangiectasia (A-T). This study reports the first A-T prenatal diagnosis performed in Spain by direct molecular analysis. The pregnant woman had a previous child suffering from A-T due to a deletion in the ATM gene. The ATM coding region was sequenced in the A-T patient and her parents. Then, a specific polymerase chain reaction (PCR) to detect the deletion was performed for prenatal diagnosis. Additionally, polymorphic HLA loci were examined in order to exclude the possible contamination by maternal DNA. In this family of Gypsy origin, we carried out a rapid molecular diagnosis of A-T. Then, a prenatal diagnosis was carried out, identifying the deletion in the fetal DNA. Additionally, we performed a population study in unrelated Spanish Gypsies and in unrelated controls, showing that the deletion described could be a hotspot in the Spanish Gypsy population. The size of the coding region and the genomic structure, together with the absence of hotspots, make the mutation screening of the ATM gene difficult. The ability to identify ATM mutations provides a tool that can be applied in confirmatory diagnosis, genetic counselling, carrier prediction and prenatal diagnosis. Copyright © 2007 John Wiley & Sons, Ltd.     

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 anaylsis 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.     

The failure of non-invasive prenatal testing due to maternal dermatomyositis

We experienced a case of a pregnant woman who failed to obtain a result from NIPT, due to the high level of total cell-free DNA. A subsequent ultrasound examination discovered that the fetus had severe intrauterine growth restriction, so the woman decided to abort the baby. At the same time, the woman developed slight swelling and tenderness of the proximal interphalangeal and meta-carpophalangeal joints. At first, these symptoms were not noticed, but, when the pregnant woman was admitted to the hospital, her laboratory tests were seriously abnormal, such as serum lactate dehydrogenase (640U/L), creatine phosphor kinase (4525U/L), kinase isoenzyme MB (170U/L), and a hydroxybutyrate dehydrogenase (398U/L). The patient had no other symptoms at this time. Misoprostol and subsequent forceps curettage were used for the induced abortion, a 167-g female fetus was aborted. Fetal skin tissue was taken for chromosomal microarray analysis (CMA) and placenta (biopsied in four places and tested as two composite samples) were taken for postnatal karyotyping to exclude a confined placental mosaicism, chromosomal microarray analysis of the fetal skin tissue revealed that the karyotype was 46, XX, karyotyping of placenta (100 cells) gave results of 46, XX, no abnormalities were detected. Ten days after induction, the patient had developed progressive symmetric muscle weakness in the proximal extremities. Physical examination revealed Gottron's sign and erythema. A manual muscle test showed weakness of the muscles (4/5) of her proximal extremities. Electromyography showed myogenic impairment. After excluding the possibility of neoplasia, the patient was diagnosed with dermatomyositis.     

Prenatal exclusion of haemophilia a and carrier testing by direct detection of a disease lesion

A novel mutation was detected in the Factor VIII gene of a sporadic case of severe haemophilia A. The lesion, a CGA → TGA transition, converts Arg 795 to Term and adequately accounts for the severe phenotype observed. PCR/direct sequencing was used to confirm the carrier status in the mother. Exclusion of haemophilia A in an at-risk pregnancy was then achieved by demonstration of the absence of this lesion in fetal DNA from a chorionic villus sample. The mutation was also detectable by chemical cleavage of mismatch (CCM), which both confirmed the prenatal diagnosis and established the carrier status of the proband's sister. This example therefore serves to illustrate the potential of direct gene analysis in sporadic cases of haemophilia A and/or in families uninformative for known RFLPs.