Haplotype analysis of oculopharyngeal muscular dystrophy (OPMD) locus in Yakutia

Oculopharyngeal muscular dystrophy (OPMD) is a hereditary neuromuscular disease with autosomal dominant and rarely with autosomal recessive inheritance types. This study included 50 patients with a clinical diagnosis of OPMD, 23 asymptomatic carriers of the mutation from 45 unrelated families, and 56 healthy relatives, as well as population samples of four ethnic groups of Yakutia: Yakuts, Evens, Evenks, Yukaghirs. It was found that the cause of OPMD development in all investigated families is the same increase in GCN repeats to 14 copies in the PABPN1 gene. The molecular structure of the (GCN)₁₄ mutant allele is (GCG)₁₀(GCA)₃GCG. The genetic variability of ten SNPs at the OPMD locus was studied in patient families and population samples. The haplotypes of OPMD were determined by a segregation analysis technique and using the EM algorithm in the groups of patients, mutation carriers, and population samples. Only one haplotype of four SNPs (ATCG) linked with the (GCN)₁₄ mutant allele was found in Yakuts and Russian patients and OPMD mutation carriers. Probably, this indicates the accumulation of mutations as a result of the founder effect.     

A new method for the analysis of age trends in CPK levels with application to Duchenne muscular dystrophy.

Measurement of serum creatine phosphokinase (CPK) is the most commonly applied test for carrier detection in Duchenne muscular dystrophy. About two thirds of all carriers have markedly elevated CPK levels. Age correction of CPK measurements would be straightforward if carriers of all ages could be unambiguously identified. Since such identification is impossible, we elaborate an indirect statistical method which is based on Haldane's theory of the balance between selection and mutation for X-linked lethals. We also apply this method to a large body of data gathered on female relatives of Duchenne muscular dystrophy patients and on controls. The results are compared with earlier partial findings.     

The Identification of Carriers in Duchenne Muscular Dystrophy

Following recurrent reports in the literature that it is possible to detect carriers of the gene for progressive muscular dystrophy (Duchenne), a survey of nine families was performed. No unequivocal results could be obtained in comparison of clinical data with circulation-time measurements or serum enzyme studies. It is concluded that carrier-labelling with respect to progressive muscular dystrophy is not yet at a satisfactory stage, although progress is being made.     

A rare subclinical or mild type of Becker muscular dystrophy caused by a single exon 48 deletion of the dystrophin gene

In the material of 227 families with Becker muscular dystrophy (BMD), we found nine non-consanguineous families with 17 male individuals carrying a rare mutation—a single exon 48 deletion of the dystrophin gene—who were affected with a very mild or subclinical form of BMD. They were usually detected thanks to accidental findings of elevated serum creatine phosphokinase (sCPK). A thorough clinical analysis of the carriers, both children (12) and adults (5), revealed in some of them muscle hypotonia (10/17) and/or very mild muscle weakness (9/17), as well as decreased tendon reflexes (6/17). Adults, apart from very mild muscle weakness and calf hypertrophy in some, had no significant abnormalities on neurological assessments and had good exercise tolerance. Parents of the children carriers of the exon 48 deletion are usually unaware of their children being affected, and possibly at risk of developing life-threatening cardiomyopathy. The same concerns the adult male carriers. Therefore, the authors postulate undertaking preventive measures such as cascade screening of the relatives of the probands. Newborn screening programmes of Duchenne muscular dystrophy (DMD)/BMD based on sCPK marked increase may be considered.     

Use of creatine kinase for detecting severe X-linked muscular dystrophy carriers.

Women thought to be at risk of being carriers of Duchenne muscular dystrophy were given "odds" against their having an affected child. These were calcuated from a combination of the genetic risk from the family history and an estimation of the biochemical risk from measuring the serum creatine kinase concentration. The women were told the actual risk estimate and it was put into perspective for them as a high, medium, or low risk. Of 25 women at high risk six have had children, all girls; the two in the medium-risk group have had no children; and the 46 women at low risk have had 19 boys and 25 girls. None of the boys has the disease. With detailed counselling most potential carriers of this disease reach decisions in child bearing that are in line with their degree of risk.     

Detection of New Paternal Dystrophin Gene Mutations in Isolated Cases of Dystrophinopathy in Females

Duchenne muscular dystrophy is one of the most common lethal monogenic disorders and is caused by dystrophin deficiency. The disease is transmitted as an X-linked recessive trait; however, recent biochemical and clinical studies have shown that many girls and women with a primary myopathy have an underlying dystrophinopathy, despite a negative family history for Duchenne dystrophy. These isolated female dystrophinopathy patients carried ambiguous diagnoses with presumed autosomal recessive inheritance (limbgirdle muscular dystrophy) prior to biochemical detection of dystrophin abnormalities in their muscle biopsy. It has been assumed that these female dystrophinopathy patients are heterozygous carriers who show preferential inactivation of the X chromosome harboring the normal dystrophin gene, although this has been shown for only a few X:autosome translocations and for two cases of discordant monozygotic twin female carriers. Here we study X-inactivation patterns of 13 female dystrophinopathy patients—10 isolated cases and 3 cases with a positive family history for Duchenne dystrophy in males. We show that all cases have skewed X-inactivation patterns in peripheral blood DNA. Of the nine isolated cases informative in our assay, eight showed inheritance of the dystrophin gene mutation from the paternal germ line. Only a single case showed maternal inheritance. The 10-fold higher incidence of paternal transmission of dystrophin gene mutations in these cases is at 30-fold variance with Bayesian predictions and gene mutation rates. Thus, our results suggest some mechanistic interaction between new dystrophin gene mutations, paternal inheritance, and skewed X inactivation. Our results provide both empirical risk data and a molecular diagnostic test method, which permit genetic counseling and prenatal diagnosis of this new category of patients.     

Prenatal cytogenetic analysis of women with high risk for genetic disorders

165 prenatal cytogenetic analyses are reported. The culture and Giemsa or quinacrine mustard (QM) staining processes are described. Karyotypes from both Giemsa and QM metaphases were analyzed. The main indications for amniocentesis were: 1)previous child with Down's syndrome (65), 2)advanced maternal age (74), 3)D/G carrier (5), 4)Duchenne muscular dystrophy (5) or 6)previous indication of other chromosomal anomaly. In the advanced maternal age group, 4 G21 and 1 E18 trisomy fetuses were detected. No chromosomal abnormalities were seen in the group referred for a previous child with Down's syndrome, although one woman was found to have a 9/13 translocation herself. Another woman with 13/14 translocation gave birth to a healthy boy with a 13/14 translocation, as predicted. Of 5 women referred for D/G translocation carriers, 1 had a fetus with a 46, X,Y,-D + t(DqGq) karyotype. Sex determination for X-linked anomalies resulted in detection of 2 Duchenne's muscular dystrophy, 1 hemophilia, 1 Norrie's syndrome, and 1 Pelizaeus-Merzbacher's syndrome.     

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.     

miRNome profiling in Duchenne muscular dystrophy; identification of asymptomatic and manifesting female carriers

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disorder that occurs due to inactivating mutations in DMD gene, leading to muscular dystrophy. Prediction of pathological complications of DMD and the identification of female carriers are important research points that aim to reduce disease burden. Herein, we describe a case of a late DMD patient and his immediate female family members, who all carry same DMD mutation and exhibited varied degrees of symptoms. In our study, we sequenced the whole miRNome in leukocytes and plasma of the family members and results were validated using real-time PCR. Our results highlighted the role of miR-409-3p, miR-424-5p, miR-144-3p as microRNAs that show correlation with the extent of severity of muscular weakness and can be used for detection of asymptomatic carriers. Cellular and circulating levels of miR-494-3p had shown significant increase in symptomatic carriers, which may indicate significant roles played by this miRNA in the onset of muscular weakness. Interestingly, circulating levels of miR-206 and miR-410-3p were significantly increased only in the severely symptomatic carrier. In conclusion, our study highlighted several miRNA species, which could be used in predicting the onset of muscle and/or neurological complications in DMD carriers.     

A method for the detection of carriers of duchenne muscular dystrophy—A preliminary report

This report of a method to detect carriers of Duchenne muscular dystrophy describes a technique of tissue autoradiography that identifies increased uptake of [³H]leucine into some individual muscle fibers in muscle biopsies. Three obligate and seven of 11 sisters of affected boys are identified as carriers in this series. This method of identification may be of value in identifying those carriers undetected by other means, but remains to be further evaluated.     

Estimates of conditional heterozygosity risks for young females in Duchenne muscular dystrophy

Using the data from daughters of known carriers and from age-paired controls, we present a method for estimating the mean and variance of creatine kinase (CK) and pyruvate kinase (PK) in pre-menarchal and early adolescent Duchenne muscular dystrophy (DMD) carriers. CK and PK means and variances were estimated for different age ranges; it is shown that among DMD carriers the levels of both enzymes decrease linearly with age. A discriminant analysis was further performed for the estimation of biochemical risks favouring the diagnosis of heterozygosity for possible young carriers. The use of this method may also be applicable for other X-linked conditions in which the detection of heterozygotes is probabilistic.     

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.     

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.     

(Na++K+)-ATPase of Duchenne muscular dystrophy erythrocyte ghosts.

Adenosine triphosphatase (ATPase) activity of erythrocyte ghosts from Duchenne muscular dystrophy (DMD) patients and carriers was stimulated by ouabain while nonmyopathic donor preparations were inhibited. Ethacrynic acid was an effective inhibitor in both DMD patients and carriers as well as in controls. However, responsiveness of the enzyme to suramin and harmaline generally differed in DMD groups from that of nonmyopathic donors. These data are consistent with the hypothesis that modified affinity of Na binding site(s) may account for some properties of the (Na⁺+K⁺)-ATPase activity of DMD erythrocytes.     

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。     

Canine muscular dystrophy: confirmation of X-linked inheritance

The genetic basis of muscular dystrophy in golden retriever dogs was investigated by means of experimental matings and cytogenetic studies. An affected male golden retriever was mated to three normal females, producing an F1 generation of six males and 14 females, all of which were clinically normal. Of six F1 females retained for breeding, all were shown to be carriers of muscular dystrophy in outcrosses to unrelated normal male dogs or in backcrosses to the affected male golden retriever. In outcrosses of carrier females, three of seven male and none of nine female offspring were affected, as expected under the X-linked recessive hypothesis. Backcrosses of F1 females to their affected sire also yielded results that are consistent with this hypothesis: 15 of 32 males and 5 of 17 females had muscular dystrophy. Cytogenetic studies of a carrier female, an affected male offspring, and a normal male sibling revealed no detectable abnormalities of the X chromosome.     

Carrier detection of Duchenne dystrophy by frequency analysis of the electromyogram

A new method for Duchenne muscular dystrophy (DMD) carrier detection based on frequency analysis of electromyograms (EMG) taken from relative force contractions shows great promise in classifying possible carriers. Nine carriers were examined and compared to nineteen normals in an attempt to define a discriminant function that would be effective in classification of possible carriers of the gene responsible for DMD. EMG data were taken at 20%, 40%, and 60% of maximum effort from the biceps brachii using a specially designed dynamometer apparatus. The apparatus was able to isolate the biceps muscle group and allowed for adjustment to accommodate different body sizes. Three signal processing methods were used to find variables in the frequency domain that would provide the best discriminant function based on the jackknife classification method. Of these methods, the most promising appears to be the high-to-low ratio method at 20% maximum contraction. When combining the high and low values found with this test in conjunction with the peak cepstrum values also found at 20% maximum contraction, discrimination was found to be 83.8% accurate. Because this classification is not based solely on definite carriers, and appears to be independent of serum creatine phosphokinase (CPK) values, it would seem that classification based on a definite carrier population could be performed with greater accuracy, especially with the addition of CPK values.     

Carrier testing of children for two X-linked diseases: a retrospective evaluation of experience and satisfaction of subjects and their mothers

Carrier testing of children for inherited disease that will not affect the health of the children themselves but of their future children is generally regarded as problematic. In this retrospective study, we determined how young women had experienced genetic carrier testing when they were children. The families of 66 young females who had been tested for carriership during childhood between 1984 and 1988, were approached. Of them, 23 young females in families affected by Duchenne muscular dystrophy, and 23 young females in families affected by hemophilia A, and their mothers, participated in our study. We used a questionnaire including multiple-choice and open-ended questions. We recorded general attitudes to testing, satisfaction with testing, degree of trust in test results, making decisions regarding testing, privacy, and opinions about age at testing. Thirty-five out of 46 of the young women tested (76%) were satisfied with carrier testing in childhood. However, the young women in whom the test results had been uncertain were statistically more often unsatisfied with the testing than those who had been found or not found to be carriers (p = 0.002). In each group, the opinions of mothers were parallel to those of their daughters. Seventy-eight percent of daughters regarded carrier testing as a family matter in which parents can make a decision. About half of those tested recalled that they had been allowed to participate in decision-making in a satisfying way. Thirty-nine out of 46 (85%) of the young women tested, and 33/46 (72%) of the mothers, suggested that carrier testing should be performed in childhood or during teenage years.     

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.     

Female transmitters of Duchenne's myopathy: detection by quantitative ultrasonography combined with blood creatine kinase

Duchenne muscular dystrophy (DMD) is a disease inherited in an X-linked recessive manner. Nothing is yet known about the gene defect involved. The problem of DMD carrier detection is very important and now without satisfactory solution. Presently the best method, for this detection, is the determination of the serum creatine-kinase (CK) level. However about 30% of DMD children's mothers have normal CK levels. In this study we associated CK determination and a new echographic method. This physical investigation is based on the muscle attenuation measurements through the evolution of the running spectral moments. The parameter expressed is the slope of the ultrasound attenuation signal (in decibels by centimetre and by megahertz; dB . cm-1 . MHz-1). We measured this parameter in vivo on the vastus medialis. The use of the chemical technique and of the physical one improves largely the detection of obligate carriers of Duchenne muscular dystrophy than when one of these methods is employed alone. One such combination recognizes 17 out of the 19 obligate carriers in our series. The difference of the results obtained by these two unrelated methods cannot be yet interpreted, but can reflect genetic heterogeneity in DMD and the inactivation of the X chromosomes.