正常中国人和DMD患者X染色体Xp~(21)区的RFLP研究

本文应用从人类X柒色体Xp~(21)区不同部位分离得到的9种DNA探针,分析了100名正常中国人,38名DMD患者及其母亲X柒色体Xp~(21)区的14个限制性位点多态性(RSP;又称限制性片段长度多态性,RFLP)。发现正常的X染色体与携带DMD基因的X染色体Xp~(21)区的RFLP频率没有明显差别;在38例DMD患者中有7例的X染色体有DNA片段缺失;在本文分析的24例患者母杀中有17例是DMD基因携带者,她们在Xp~(21)区的RFLP均存在杂合的多态性,因此可以应用RFLP连锁分析对这些家系进行DMD的产前诊断。     

Linkage analysis of two cloned DNA sequences flanking the Duchenne muscular dystrophy locus on the short arm of the human X chromosome.

The inheritance of two restriction fragment length polymorphisms (RFLPs) on the short arm of the human X chromosome has been studied relative to Duchenne muscular dystrophy. This provides a partial genetic map of the short arm of the human X chromosome between Xp110 and Xp223. The data were derived from the segregation between a RFLP located at Xp21-Xp223, the DMD locus, and a RFLP located at Xp110-Xp113. The genetic distance from Xp110 to Xp223 was found to be approximately 40 centimorgans (cM). This provides experimental confirmation that 1cM corresponds to approximately 1,000 kilobase pairs of DNA for this region of the human X chromosome. Our data confirm that the DMD mutation lies between Xp223 and Xp110. The availability of flanking probes surrounding the DMD locus will assist in the ordering of further DNA sequences relative to the mutation.     

Isolation of probes detecting restriction fragment length polymorphisms from X chromosome-specific libraries: potential use for diagnosis of Duchenne muscular dystrophy

Summary We have isolated 23 human X chromosome-specific DNA fragments from libraries, prepared from flow-sorted X chromosomes. To increase diagnostic potential for X-linked genetic disorders, including Duchenne muscular dystrophy (DMD), the fragments were tested for restriction fragment length polymorphisms (RFLPs) with six restriction enzymes. All fragments were regionally mapped to segments of the X chromosome with a panel of somatic cell hybrids and with human cell lines carrying unbalanced chromosomal abnormalities. Two of the isolated probes detected a high frequency RFLP. One, 754, maps between Xp11.3 and Xp21 and detects a PstI polymorphism with an allele frequency of 0.38. The other, 782, maps between Xp22.2 and Xp22.3 and reveals an EcoRI polymorphism with an allele frequency of 0.40. According to a pilot linkage study of families at risk for Duchenne muscular dystrophy, 754 gives a maximum Lod score of 7.6 at a recombination fraction of 0.03. Probe 782 lies telomeric to DMD with a maximum Lod score of 2.2 at a recombination fraction of 0.17. Using our X-chromosomal probes and a set of autosomal probes, isolated and examined in an identical way, we found a significantly lower RFLP frequency for the X chromosome as compared to the autosomes.     

The Metabolome in Finnish Carriers of the MYBPC3-Q1061X Mutation for Hypertrophic Cardiomyopathy

Aims

Mutations in the cardiac myosin-binding protein C gene (MYBPC3) are the most common genetic cause of hypertrophic cardiomyopathy (HCM) worldwide. The molecular mechanisms leading to HCM are poorly understood. We investigated the metabolic profiles of mutation carriers with the HCM-causing MYBPC3-Q1061X mutation with and without left ventricular hypertrophy (LVH) and non-affected relatives, and the association of the metabolome to the echocardiographic parameters.

Methods and Results

34 hypertrophic subjects carrying the MYBPC3-Q1061X mutation, 19 non-hypertrophic mutation carriers and 20 relatives with neither mutation nor hypertrophy were examined using comprehensive echocardiography. Plasma was analyzed for molecular lipids and polar metabolites using two metabolomics platforms. Concentrations of branched chain amino acids, triglycerides and ether phospholipids were increased in mutation carriers with hypertrophy as compared to controls and non-hypertrophic mutation carriers, and correlated with echocardiographic LVH and signs of diastolic and systolic dysfunction in subjects with the MYBPC3-Q1061X mutation.

Conclusions

Our study implicates the potential role of branched chain amino acids, triglycerides and ether phospholipids in HCM, as well as suggests an association of these metabolites with remodeling and dysfunction of the left ventricle.     

Duchenne muscular dystrophy due to familial Xp21 deletion detectable by DNA analysis and flow cytometry

Summary We report two male cousins with Duchenne muscular dystrophy (DMD) in whom cytogenetic studies have shown a small interstitial deletion at Xp21. The lesion is readily detectable in patients and carriers by flow cytometry which indicates that approximately 6000 kb of DNA are deleted in each case. The DNA markers OTC, C7, and B24 are present in the deleted X chromosome but 87-8, 87-1, and 754 are absent. Despite apparently identical deletions one affected boy has profound mental handicap while the other is only mildly retarded. The results confirm the assignment of familial DMD to Xp21 and illustrate the value of flow cytometry in improving the precision of chromosome analysis. We have also undertaken flow cytometry in a cell line from a previously reported DMD patient with a de novo Xp21 deletion who had, in addition, chronic granulomatous disease, retinitis pigmentosa, and the McLeod syndrome. The results indicate that the amount of DNA deleted from the X is similar in both families despite the striking differences in phenotype.     

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.     

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.     

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.     

The Xq22 inversion breakpoint interrupted a novel Ras-like GTPase gene in a patient with Duchenne muscular dystrophy and profound mental retardation

A male patient with profound mental retardation, athetosis, nystagmus, and severe congenital hypotonia (Duchenne muscular dystrophy [DMD]) was previously shown to carry a pericentric inversion of the X chromosome, 46,Y,inv(X)(p21.2q22.2). His mother carried this inversion on one X allele. The patient's condition was originally misdiagnosed as cerebral palsy, and only later was it diagnosed as DMD. Because the DMD gene is located at Xp21.2, which is one breakpoint of the inv(X), and because its defects are rarely associated with severe mental retardation, the other clinical features of this patient were deemed likely to be associated with the opposite breakpoint at Xq22. Our precise molecular-cytogenetic characterization of both breakpoints revealed three catastrophic genetic events that had probably influenced neuromuscular and cognitive development: deletion of part of the DMD gene at Xp21.2, duplication of the human proteolipid protein gene (PLP) at Xq22.2, and disruption of a novel gene. The latter sequence, showing a high degree of homology to the Sec4 gene of yeast, encoded a putative small guanine-protein, Ras-like GTPase that we have termed "RLGP." Immunocytochemistry located RLGP at mitochondria. We speculate that disruption of RLGP was responsible for the patient's profound mental retardation.     

Kreatin-Phosphokinase-Aktivität im Serum bei Konduktorinnen der progressive Muskeldystrophie vom Typ Duchenne

Zusammenfassung Bei 148 weiblichen Verwandten von 33 Patienten mit Duchennescher Muskeldystrophie in 21 Sippen sowie bei 25 Kontrollpersonen ist die Kreatin-Phosphokinase bestimmt worden. Die Getesteten befanden sich im Alter zwischen 6 Monaten und 79 Jahren. Erhöhte CPK-Werte fanden wir bei 7 (88%) von 8 sicheren und wahrscheinlichen Genträgerinnen.Von den 30 möglichen Konduktorinnen der Gruppe A — bei denen das statistische Risiko der Heterozygotie bekannt ist — wiesen 17 (52%) über die Norm erhöhte CPK-Werte auf. Die Zahl der als Heterozygote identifizierten Personen entsprach der statistischen Erwartung von 13. Von den 14 Müttern isoliert erkrankter Patienten wurden bei 10 (71,5%) erhöhte CPK-Aktivitäten nachgewiesen.In der Gruppe B der möglichen Trägerinnen, bei denen das statistische Risiko, heterozygot zu sein, nicht bekannt ist, konnten bei insgesamt 110 weiblichen Verwandten bei 32 Personen (29%) erhöhte CPK-Aktivitäten gemessen werden.In den 4 Sippen der genetisch sicheren Konduktorinnen können wir auf Grund unserer Befunde nicht bestimmen, zu welchem Zeitpunkt die Neumutation auftrat. Das gleiche gilt für weitere 9 Sippen, während bei den restlichen 8 der Mutationszeitpunkt diskutiert wird.Die CPK-Bestimmung zur Identifizierung von Heterozygoten hat sich als bester Indicator erwiesen. Die ca. 25% falsch-negativen Werte sind durch Mehrfachbestimmungen zu mindern. Mögliche Ursachen der falsch-negativen Werte sind intraindividuelle Schwankungen, meßtechnische Fehler und die Mosaikstruktur heterozygoter Gene gemäß der Lyon-Hypothese.

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Serum creatine-phosphokinase activity in female carriers of muscular dystrophy (type Duchenne)

Summary Serum creatine-phosphokinase activity has been investigated in 148 female relatives of 33 patients with progressive muscular dystrophy (type Duchenne) and in 25 controls. The age of the relatives ranged from 6 months to 79 years. Elevated creatine kinase activity was found in 7 (88%) of 8 definite and probable carriers. Among 30 possible carriers in whom the risk of heterozygosity could be calculated, 17 subjects were identified as carriers, in close agreement with the expectation of 13. — Among 110 other possible carriers, in whom the risk of heterozygosity could not be determined, 32 carriers were identified. Of the 14 mothers of sporadic cases, included in this group, high CK activity was found in 10. — In the 4 families of the definite carriers it was not possible to determine at which time the new mutation took place. In 8 families of possible carriers data supporting the appearance of the mutation in some subjects are discussed. — For genetic prognosis, an elevated CK value in a relative of a Duchenne patient is regarded as reliable evidence of heterozygosity, but if the CK value is within the normal range there is an appreciable risk that it is a false negative and that the subject is actually a carrier. Possible causes of false negative observations are technical difficulties, decline of CK levels with increasing age, heterozygote variability consequent to lyonization of an X-chromosome, and genetic heterogeneity.

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Benutzte Abkürzungen ADP Adenosindiphosphat - ADK Adenylatkinase (Myokinase) - ALD Aldolase - ATP Adenosin-5-Triphosphat - CPK Kreatinphosphatkinase - EDTA Äthylendiamintetraessigsäure - GOT Glutamat-Oxalacetat-Transaminase - GPT Glutamat-Pyruvat-Transaminase - GSH reduziertes Glutathion - LDH Lactatdehydrogenase - MgSO₄ Magnesiumsulfat - NAD Nicotinadenindinucleotid - NADH Nicotin-adenindinucleotid, reduzierte Form - NaOH Natronlauge - PEP Phosphoenolpyruvat - PK Pyruvatkinase - PMD progressive Muskeldystrophie - P Phosphat - TRAP Triäthanolaminpuffer     

Segregation analysis of a marker localised Xp21.2-Xp21.3 in Duchenne and Becker muscular dystrophy families

Summary A DNA marker C7, localised Xp21.1-Xp21.3, has been studied in kindreds segregating for Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). In DMD families four crossovers were observed in 38 informative meioses between C7 and the DMD locus (=0.12, z _max=+2.72). In BMD families no recombinants were observed in the 16 informative meioses studied. These data are consistent with the localisation of the mutations in these disorders being in the same region of Xp21.Studies in families also segregating for the DNA marker 754 support the previously reported physical order of these loci as X centromere-754-DMD-BMD-C7-X telomere. A recombination fraction of 0.11 (z _max=+5.58) was found between DMD-754 by combining our previously published data with the data presented here. C7 and 754 thus provide good bridging markers for the diagnosis of DMD and BMD.     

Localization of the McLeod locus (XK) within Xp21 by deletion analysis.

The McLeod phenotype is an X-linked, recessive disorder in which the red blood cells demonstrate acanthocytic morphology and weakened antigenicity in the Kell blood group system. The phenotype is associated with a reduction of in vivo red cell survival, but the permanent hemolytic state is usually compensated by erythropoietic hyperplasia. The McLeod phenotype is accompanied by either a subclinical myopathy and elevated creatine kinase (CK) or X-linked chronic granulomatous disease (CGD). Seven males with the McLeod red-blood-cell phenotype and associated myopathy but not CGD, one male with the McLeod phenotype associated with CGD, and two males known to possess large deletions of the Duchenne muscular dystrophy (DMD) locus were studied. DNA isolated from each patient was screened for the presence or absence of various cloned sequences located in the Xp21 region of the human X chromosome. Two of the seven males who have only the McLeod phenotype and are cousins exhibit deletions for four Xp21 cloned fragments but are not deleted for any portion of either the CGD or the DMD loci. Comparison of the cloned segments absent from these two McLeod cousins with those absent from the two DMD boys and the CGD/McLeod patient leads to the submapping of various cloned DNA segments within the Xp21 region. The results place the locus for the McLeod phenotype within a 500-kb interval distal from the CGD locus toward the DMD locus.     

Estimation of the male to female ratio of mutation rates from the segregation of X-chromosomal DNA haplotypes in Duchenne muscular dystrophy families

Summary A novel procedure is presented to estimate the ratio of male to female mutation rates for Duchenne muscular dystrophy (DMD). X-specific restriction fragment length polymorphisms are used to establish DNA haplotypes in three-generation DMD families. From the proportion of DMD patients who have inherited their maternal grandfather's X chromosome, the ratio of mutation rates can be calculated. In contrast to classical methods, the proposed procedure is not restricted to sporadic or familiar cases nor is any information on the carrier status of female relatives required.     

Turner's syndrome and Duchenne muscular dystrophy in a girl with an X; autosome translocation

A balanced de novo (X;9) translocation was observed in a patient with progressive muscular dystrophy of Duchenne's type (DMD), Turner's syndrome, epilepsy and mental retardation. The involvement of the paternal X is suggested. The assignment of the gene locus for DMD is confirmed on Xp21.     

Molecular analysis of the Duchenne muscular dystrophy region using pulsed field gel electrophoresis

Genetic and molecular studies show that the Duchenne muscular dystrophy (DMD) locus at Xp21 is large and complex. We have analyzed this region using pulsed field gel electrophoresis (PFGE) and have determined physical distances between Xp21 probes. The sum of the sizes of the Sfil restriction fragments detected by these probes is greater than 4000 kb. The deletion endpoints in two DMD patients were detected by observing changes in these restriction fragments. In addition, the Xp21 breakpoint for the X;1 translocation in an affected female was mapped. These results demonstrate the applicability of PFGE for analysis of Xp21, and should facilitate the mapping of other translocations and deletions in this region, some of which lead to glycerol kinase deficiency and adrenal hypoplasia as well as DMD.     

Exclusion mapping of the gene for X-linked neural tube defects in an Icelandic family

Various polymorphic markers with a random distribution along the X chromosome were used in a linkage analysis performed on a family with apparently Xlinked recessive inheritance of neural tube defects (NTD). The lod score values were used to generate an exclusion map of the X chromosome; this showed that the responsible gene was probably not located in the middle part of Xp or in the distal region of Xq. A further refining of these results was achieved by haplotype analysis, which indicated that the gene for X-linked NTD was located either within Xp21.1-pter, distal from the DMD locus, or in the region Xq12–q24 between DXS106 and DXS424. Multipoint linkage analysis revealed that the likelihood for gene location is highest for the region on Xp. The region Xq26–q28, which has syntenic homology with the segment of the murine X chromosome carrying the locus for bent tail (Bn), a mouse model for X-linked NTD, is excluded as the location for the gene underlying X-linked NTD in the present family. Thus, the human homologue of the Bn gene and the present defective gene are not identical, suggesting that more than one gene on the X chromosome plays a role in the development of the neural tube.     

Isolation of a conserved sequence deleted in Duchenne muscular dystrophy patients.

We have isolated a DNA sequence (HIP25) by subtraction- hybridisation which is deleted in a number of Duchenne muscular dystrophy (DMD) patients. HIP25 is conserved in evolution and hybridises to human fetal and adult muscle mRNA. HIP25 is absent in human fetal fibroblast mRNA. Physical mapping data localise this sequence within Xp21 between the breakpoints of X;autosome translocations found in two females suffering from the disease. HIP25 is a candidate exon sequence for the basic defect in DMD boys deleted at this locus.     

Birth and population prevalence of Duchenne muscular dystrophy in the Netherlands

Summary Mutations causing Duchenne muscular dystrophy (DMD) have a short survival. Therefore, birth and population prevalence are maintained by new mutations. The present inventory was made to estimate the birth and population prevalence rates of DMD in the Netherlands. Seven methods of case identification were used. Data on 496 definite, probable or possible DMD patients born since 1961, or alive on January 1, 1983, were obtained. Several methods gave an estimated ascertainment of more than 95%. The prevalence rate at birth of DMD was estimated at 23.7×10^–5 (14215) male live births (MLB) yearly. The prevalence rate in the male population on January 1, 1983 was 5.4×10^–5 (118496). About 1% of the males in this study may have autosomal recessive Duchenne-like muscular dystrophy. Until now there has been no convincing evidence for geographic differences in DMD prevalence at birth. A list of frequency studies of Duchenne muscular dystrophy is included. The DMD mutation rate calculated by the indirect method is 7.9×10^–5 genes per generation. However, this may well be an over-estimate, as this method does not account for germline mosaicism. Using a modified sex ratio method the proportion of sporadic DMD among all cases was estimated to be 0.106 (range 0–0.332). High frequency of germline mosaicism in DMD is a likely cause for the apparent lack of sporadic cases as found in previous studies, if mutation rates in male and female gametes are equal. Therefore, methods for estimating the proportion of new mutants in DMD should take germline mosaicism into account. The modified sex ratio method allows incorporation of data on germline mosaicism if available.