Characterization of patients with glycerol kinase deficiency utilizing cDNA probes for the Duchenne muscular dystrophy locus

Summary Genomic DNA from five previously unreported patients with glycerol kinase deficiency (GKD), dystrophic myopathy, and adrenal insufficiency were studied with genomic probes and cDNA probes for the Duchenne muscular dystrophy (DMD) locus. These individuals, together with those reported by ourselves and others, show that patients with a contigous gene syndrome involving the DMD, GK, and adrenal hypoplasia congenita (AHC) loci have a broader distribution of microdeletion breakpoints than those observed among patients with classical DMD. This study demonstrates the use of the DMD cDNA probes to delineate the centromeric deletion breakpoints for patients with Xp21 microdeletions extending beyond the DMD locus. It also shows the practical diagnostic application of the DMD cDNA probes when the diagnosis of GKD is entertained in a patient with known DMD and only DNA is available for study.     

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。     

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.     

Familial deletion of Xp21.2 with glycerol kinase deficiency and congenital adrenal hypoplasia

Summary Congenital adrenal hypoplasia (CAH) and glycerol kinase deficiency (GKD) were diagnosed in a male during the neonatal period. On prometaphase chromosomes there was an interstitial deletion involving Xp21.2 and possibly Xp21.3 in the propositus and his mother. Duchenne muscular dystrophy (DMD) was excluded on the basis of normal serum creatine kinase and a muscle biopsy. Molecular hybridization of DNA from the propositus with 11 probes covering Xp21, including the DMD locus, was normal. In situ hybridization with the probe pERT87.15 showed a normal signal at the expected site indicating that the DMD locus was preserved and not translocated. This suggests that the DMD locus is located at the most proximal part of the sub-band Xp21.2 or in Xp21.1, and that the DXS68 (probe L1) is far from it on the distal flanking DNA.     

Identification of new markers in Xp21 between DXS28 (C7) and DMD

Characterization of Xp21 distal to Duchenne muscular dystrophy (DMD) in the region containing the genes for adrenal hypoplasia congenita (AHC) and glycerol kinase deficiency (GKD) has been limited due to a paucity of probes. Two probes were localized between DXS28 (C7) and AHC, the yeast artificial chromosome insert YHX39 (DXS727) and the polymorphic phage clone QST59 (DXS319). A genomic clone, FT1 (DXS726), 3' to DMD, was also characterized. Portions of the three probes were sequenced and primer pairs were generated to amplify a sequence-tagged site within each probe. Amplification of DNA from patients confirmed the deletion results obtained by Southern blot analysis, and these three sequence-tagged sites were successfully combined for triplex PCR. In addition to facilitating molecular genetic diagnosis in Xp21, these probes can be used to identify additional YACs and other probes to further increase the genomic information and diagnostic capabilities in this region.     

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.     

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.     

Localization and cloning of Xp21 deletion breakpoints involved in muscular dystrophy

Summary Twenty-nine deletion breakpoints were mapped in 220 kb of the DXS164 locus relative to potential exons of the Duchenne and Becker muscular dystrophy gene. Four deletion junction fragments were isolated to acquire outlying Xp21 loci on both the terminal and centromere side of the DXS164 locus. The junction loci were used for chromosome walking, searches for DNA polymorphisms, and mapping against deletion and translocation breakpoints. Forty-four unrelated deletions were analyzed using the junction loci as hybridization probes to map the endpoints between cloned Xp21 loci. DNA polymorphisms from the DXS164 and junction loci were used to follow the segregation of a mutation in a family that represents a recombinant. Both the physical and genetic data point to a very large size for this X-linked muscular dystrophy locus.     

DNA deletions in mild and severe Becker muscular dystrophy

Summary The DNA of 33 patients diagnosed as suffering from Becker muscular dystrophy (BMD) has been probed with cloned DNA sequences from Xp21, known to reveal DNA deletions in patients suffering from the more severe Duchenne muscular dystrophy (DMD). Two BMD cases showed clear deletions. A third case gave aberrant band sizes, which further analysis showed to be caused by a small deletion. This suggests that deletions in DXS164 occur approximately as frequently in BMD as they do in DMD. Of the two cases showing large deletions, one is at the severe end of the Becker clinical spectrum, whilst the other is a classical Becker-type dystrophy. The fact that loci defined by probes commonly deleted in classical DMD patients are also deleted in BMD patients of varying severity is strong additional evidence that these disorders are allelic, and further justifies the use of probes with defined linkage relationships to DMD also being used for counselling in BMD families.     

The use of field-inversion gel electrophoresis for deletion detection in Duchenne muscular dystrophy.

Deletion is a common cause of Duchenne muscular dystrophy (DMD). Field-inversion gel electrophoresis, in conjunction with Southern blot hybridization, was used to detect large SfiI DNA fragments in the DMD locus. Two unrelated boys with DMD were found to have abnormal sized DNA fragments resulting from deletions. Some of the female relatives of these patients were also shown by this method to have deletions in the DMD locus.     

Mapping of Xp21 translocation breakpoints in and around the DMD gene by pulsed field gel electrophoresis

Balanced translocations with a breakpoint in the Xp21 region are likely to disrupt the giant Duchenne muscular dystrophy (DMD) locus and can be demonstrated in females suffering from the disease. Pulsed field gel electrophoresis allows the positioning of these breakpoints by detecting junction fragments on the derived chromosomes; DNA probes hybridizing to these fragments may be located as many as several hundred kilobases away from the breakpoints. By using this approach, 11 translocation breakpoints from the Xp21 region have been analyzed. The localization of three previously examined breakpoints was confirmed. Six other breakpoints, including a breakpoint flanking the DMD gene and not associated with the DMD phenotype, could be positioned relative to SfiI sites on a 3.5-Mb restriction map of the region.     

Brother/sister pairs affected with early-onset, progressive muscular dystrophy: molecular studies reveal etiologic heterogeneity.

An autosomal recessive (AR) form of muscular dystrophy that clinically resembles Duchenne/Becker types exists, but its frequency is unknown. We have studied three unrelated affected brother/sister pairs and their families for deletions and polymorphisms with the entire dystrophin cDNA and other DNA probes from the Xp21 region to test for involvement of the DMD locus. In family 1 a large intragenic deletion was found in the affected male. The affected sister was heterozygous for this deletion, but the mother was not, implying germinal mosaicism. In family 2, no deletion was detected in the affected male. RFLP analysis revealed that the affected male and an unaffected sister shared a complete Xp21 haplotype while the affected sister had inherited a recombinant Xp21 region resulting from a crossover between pERT 87-15 and J-Bir. Only the 5' region of the dystrophin gene was shared with the affected boy. X-inactivation studies using a polymorphism in the 5'-flanking region of the HPRT gene, in conjunction with methylation-sensitive enzymes, revealed random X inactivation in the affected girl's leukocytes. In a muscle biopsy from the affected male, the dystrophin protein was present in normal amount and size. Family 3 was informative for four RFLPs detected with dystrophin cDNA probes which span the entire gene. The affected male was found to share the complete dystrophin RFLP haplotype with his unaffected brother, while his affected sister had inherited the other maternal haplotype. It is concluded that the clinical presentation of early-onset, progressive muscular dystrophy in a male and in his karyotypically normal sister can be caused by mutations at different loci. While in family 1 a deletion in the dystrophin gene is responsible, this gene does not appear to be involved in families 2 and 3.     

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.     

Further studies of gene deletions that cause Duchenne and Becker muscular dystrophies

Fetal muscle cDNA clones covering at least 11.4 kb of the Duchenne muscular dystrophy (DMD) gene sequence were used to identify a deletion-prone region in DNA from DMD and Becker muscular dystrophy (BMD) patients. Of 36 BMD cases, 17 (47%) had deletions and all of the deletions began in the same intron of the gene. Of 107 DMD patients, 27 (25%) were deleted for this region, and 19 deletions originate in the same intron. Using a cDNA probe for an adjacent region of the gene, 32 new deletions were detected in DMD patients (total 44%). No new BMD deletions were detected. The DMD deletions were very heterogeneous. Thus two cDNA probes covering 2.4 kb could detect 53% of these deletions. Considering the whole locus, DMD and BMD are caused by a deletion of the gene sequence in at least 67% of cases.     

Molecular analysis of Duchenne and Becker muscular dystrophy

Duchenne muscular dystrophy (DMD) is a progressive and lethal neuromuscular disorder caused by a defective gene on the X chromosome. There is no effective treatment and the biochemical defect is yet unknown. Mapping of the DMD locus to band Xp21 in the short arm of the X chromosome has given rise to strategies to clone the gene from its known location in the chromosome. Two cloning strategies have led to the isolation of a gene that is the largest of any yet described. Portions of the gene are deleted in about 8% of affected males, and rare translocations that disrupt the gene cause the disease in females. The isolation of expressed sequences from the DMD locus will undoubtedly lead to isolation of the gene product and ultimately to an understanding of the basic defect. In the meantime, DNA probes from the DMD locus provide a new and accurate approach for carrier identification and prenatal diagnosis of this dreaded disease.     

Xp21 contiguous gene syndromes: deletion quantitation with bivariate flow karyotyping allows mapping of patient breakpoints.

Bivariate flow karyotyping was used to estimate the deletion sizes for a series of patients with Xp21 contiguous gene syndromes. The deletion estimates were used to develop an approximate scale for the genomic map in Xp21. The bivariate flow karyotype results were compared with clinical and molecular genetic information on the extent of the patients' deletions, and these various types of data were consistent. The resulting map spans > 15 Mb, from the telomeric interval between DXS41 (99-6) and DXS68 (L1-4) to a position centromeric to the ornithine transcarbamylase locus. The deletion sizing was considered to be accurate to +/- 1 Mb. The map provides information on the relative localization of genes and markers within this region. For example, the map suggests that the adrenal hypoplasia congenita and glycerol kinase genes are physically close to each other, are within 1-2 Mb of the telomeric end of the Duchenne muscular dystrophy (DMD) gene, and are nearer to the DMD locus than to the more distal marker DXS28 (C7). Information of this type is useful in developing genomic strategies for positional cloning in Xp21. These investigations demonstrate that the DNA from patients with Xp21 contiguous gene syndromes can be valuable reagents, not only for ordering loci and markers but also for providing an approximate scale to the map of the Xp21 region surrounding DMD.     

Duchenne and Becker muscular dystrophy mutations: analysis using 2.6 kb of muscle cDNA from the 5'' end of the gene.

We have isolated overlapping human fetal muscle cDNAs encompassing 2.6kb which are localised very close to the 5' end of the Duchenne muscular dystrophy (DMD) gene. Using DNA from patients with deletions of previously reported genomic probes, we have mapped the exons across the region. Investigation of deletions in both DMD and Becker muscular dystrophy (BMD) patients shows the deletions to be present in 10% of cases and heterogeneous.     

Molecular and phenotypic analysis of patients with deletions within the deletion-rich region of the Duchenne muscular dystrophy (DMD) gene

Eighty unrelated individuals with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) were found to have deletions in the major deletion-rich region of the DMD locus. This region includes the last five exons detected by cDNA5b-7, all exons detected by cDNA8, and the first two exons detected by cDNA9. These 80 individuals account for approximately 75% of 109 deletions of the gene, detected among 181 patients analyzed with the entire dystrophin cDNA. Endpoints for many of these deletions were further characterized using two genomic probes, p20 (DXS269; Wapenaar et al.) and GMGX11 (DXS239; present paper). Clinical findings are presented for all 80 patients allowing a correlation of phenotypic severity with the genotype. Thirty-eight independent patients were old enough to be classified as DMD, BMD, or intermediate phenotype and had deletions of exons with sequenced intron/exon boundaries. Of these, eight BMD patients and one intermediate patient had gene deletions predicted to leave the reading frame intact, while 21 DMD patients, 7 intermediate patients, and 1 BMD patient had gene deletions predicted to disrupt the reading frame. Thus, with two exceptions, frameshift deletions of the gene resulted in more severe phenotype than did in-frame deletions. This is in agreement with recent findings by Baumbach et al. and Koenig et al. but is in contrast to findings, by Malhotra et al., at the 5' end of the gene.     

Regional localisation of X chromosome short arm probes

Summary Nine human X chromosome-specific clones have been isolated by screening an X-chromosomal genomic library with fetal muscle cDNA. Five of the clones have been localised to the short arm and four to the long arm. The short arm probes have been regionally assigned using a panel of somatic cell hybrids. They have been mapped further using a series of DNA samples from male patients with different deletions of the region Xp21, and having complex phenotypes including Duchenne muscular dystrophy. The use of these probes in the mapping of the short arm of the X chromosome is discussed.     

Fluorescence in situ hybridization establishes the order cen-DXS28(C7)-DXS67(B24)-DXS68(L1)-tel in human chromosome Xp21.3.

We report here on the order of three DNA markers, C7, B24, and L1, based on the arrangement of their fluorescently labeled hybridization sites in interphase cell nuclei. The three markers map distal to the Duchenne muscular dystrophy (DMD), glycerol kinase deficiency (GKD), and adrenal hypoplasia (AHC) loci on human chromosome Xp21.3. Their order has been a matter of controversy. In interphase chromatin, B24 maps between C7 and L1. We estimate from interphase distance that C7 and L1 are 300-500 kb apart. When the three markers are hybridized to interphase cells of Nijmegen1, a patient with DMD, GKD, and AHC, only C7 appears to be deleted, rather than both C7 and L1, as had been reported elsewhere. C7 is also the only one of the three markers deleted in several other DMD patients studied by others. The deletion results indicate that C7 is the most proximal of the three markers and allow the trio of ordered probes to be oriented on the chromosome: cen-C7(DXS28)-B24(DXS67)-L1(DXS68)-tel.