Characterisation of a Xp21 microdeletion syndrome in a 2-year-old boy with muscular dystrophy,glycerol kinase deficiency and adrenal hypoplasia congenita

Summary We report a 2-year-old boy with Duchenne muscular dystrophy (DMD), glycerol kinase deficiency (GK) and adrenal hypoplasia congenita (AHC). At three weeks of age, the patient was hospitalized for the first time with symptoms of hypotone dehydration because of AHC, At present, he shows severe muscular hypotonia and developmental delay. The patient and his family were referred to us for prenatal diagnosis and carrier testing in the mother of the patient and the mother's sister, respectively. The patient's DNA was examined by Southern blot and polymerase chain reaction analyses, using cDNA and genomic probes within and around the dystrophin (DYS) locus. A deletion was revealed, spanning DXS28, the whole dystrophin locus, DXS84 and DXS148, whereas DXS67, DXS68 (pter) and OTC (cen) were found to be retained. The cytogenetically visible microdeletion was also seen in the patient's mother, but not in the mother's sister or the patient's maternal grandmother. Our findings support the locus order pter-DXS67-DXS68-DXS28-AHC-GK-DMD-cen.     

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.     

Deletion mapping of Aland Island eye disease to Xp21 between DXS67 (B24) and Duchenne muscular dystrophy.

Aland Island Eye Disease (AIED) is an X-linked form of ocular hypopigmentation--also known as Forsius-Eriksson, or type 2, ocular albinism--in which affected males demonstrate subnormal visual acuity, protanomalous red-green colorblindness, axial myopia, astigmatism, hypoplasia of the fovea, and hypopigmentation of the fundus. A patient has previously been described who, in addition to AIED, manifested a contiguous gene syndrome which included congenital adrenal hypoplasia (AHC), glycerol kinase deficiency (GKD), and Duchenne muscular dystrophy (DMD). In the present paper report we report the molecular genetic analysis of his deletion. Initially, multiplex polymerase-chain-reaction amplification was used to screen for a DMD-locus deletion which was then further characterized, using DMD cDNA and genomic probes, via Southern blot analysis. The deletion includes the region encompassed by probes C7 (DXS28) and DMD cDNA 8. Probes B24 (DXS67) and DMD cDNA 5b-7 show normal hybridization patterns and appear to flank the deletion, while the DMD cDNA 8 detects a junction fragment. Molecular genetic techniques have mapped the deletion in this patient to the subbands Xp21.3-21.2, between DXS67 and DMD.     

Deletion mutations in Duchenne muscular dystrophy (DMD) in Western Saudi children

Duchenne and Becker muscular dystrophy (DMD and BMD) are caused, in the majority of cases, by deletions in the dystrophin gene (DMD). The disease is an X-linked neuromuscular diseases typically caused by disrupting (DMD) or non-disrupting (BMD) the reading frame in the dystrophin (DMD) gene. In the present study, amplifications of the genomic DNAs of unrelated 15 Saudi DMD males were carried out using multiplex polymerase chain reaction (PCR) for nine-hotspot regions of exons 4, 8, 12, 17, 19, 44, 45, 48 and 51. We detected six Saudi patients having deletions in a frequency of 40%. The frequency of deletions in exon 51 (20%) was the most common deletion frequently associated with our Saudi sample males. Exons 19, 45, and 48 were present in a frequency of 6.7% each. All deletions were recognized as an individual exonic deletions, while no gross deletion where detected. Finally, the molecular deletions in the Saudi males was expected to be characterized by a moderate frequency among different populations due to the geographical KSA region, which it is in the crossroad of intense migrations and admixture of people coming from continental Asia, Africa, and even Europe. In conclusion, attempts to include an extra DNA samples might reflect a valid vision of the deletions within the high frequency deletion regions (HFDR’s) in the DMD gene mutations in KSA.     

Development of additional RFLP probes near the locus for Duchenne muscular dystrophy by cosmid cloning of the DXS84 (754) locus

Summary We have isolated 70 kb of sequences surrounding probe 754 (DXS84), linked with Duchenne muscular dystrophy. In addition to the original PstI RFLP detected by 754, BglII and EcoRI RFLPs were detected with the single copy subclone 754.11 and a HindIII RFLP with the subclone 754.6. The BglII and HindIII and HindIII RFLPs both have minor allele frequencies of 40%, as in PstI polymorphism. The EcoRI polymorphism has a minor allele frequency of 23%. Since a linkage disequilibrium is observed between these RFLPs (P<0.001), the BglII and the HindIII RFLPs do not contribute to the heterozygosity. However, the minor allele of the EcoRI RFLP segregates exclusively with the major haplotype of the PstI-BglII-HindIII complex, and consequently 47% of the homozygotes for the haplotype become heterozygous. As a result, the overal heterozygote frequency of the DXS84 locus increases from 50% to 65%.     

Isolation of a random cosmid clone,cX5, which defines a new polymorphic locus DXS148 near the locus for Duchenne muscular dystrophy

Summary We have isolated a random cosmid cX5 (DXS148), which maps into a small Xp21 deletion associated with Duchenne muscular dystrophy (DMD), chronic granulomatous disease (CGD), retinitis pigmentosa (RP) and McLeod syndrome. cX5 maps proximally outside several other deletions associated with DMD, glycerol kinase deficiency (GK) and adrenal hypoplasia (AHC). The following order of loci is proposed: centromere-OTC-cX5 (DXS148)-754 (DXS84)-PERT87 (DXS164)/DMD-telomere. A subclone cX5.7, isolated from this cosmid, identifies an MspI RFLP, with a minor allele frequency of 35%. This probe forms an important adjunct to the existing RFLPs for family studies in Duchenne muscular dystrophy.     

DXS28 (C7) maps centromeric to DXS68 (L1-4) and DXS67 (B24) by deletion analysis

Complex glycerol kinase deficiency (CGKD) is a contiguous gene syndrome consisting of glycerol kinase deficiency together with Duchenne muscular dystrophy (DMD), congenital adrenal hypoplasia, and/or Aland Island eye disease. Deletion mapping of genomic DNA from patients with CGKD was carried out and allowed definitive ordering of loci DXS28 (C7), DXS68 (L1-4), and DXS67 (B24). Most reports have placed DXS68 centromeric to DXS28 and DXS67 on the basis of the initial mapping of the Iowa patient 3, but others have presented evidence consistent with the placement of DXS28 telomeric to DXS68 and DXS67. Through the use of DNA from CGKD patients with a variety of genomic deletions, this controversy is resolved and the order Xcen...DMD-DXS28-DXS68-DXS67...pter is definitively demonstrated.     

Definitive localization of Becker muscular dystrophy in Xp by linkage to a cluster of DNA polymorphisms (DXS43 and DXS9)

Summary A study of linkage between Becker muscular dystrophy and four X chromosome-specific DNA polymorphisms in 17 kindreds has indicated that this gene is located in Xp, as already anticipated by single pedigree analysis. In particular the DXS43 and DXS9 loci, identified by probes D2 and RC8, respectively, are closely linked to each other and are both located at approximately 15 cM from the Becker locus. These linkage data, together with the previously established linkage between Becker and the DXS7 locus identified by probe L 1.28, indicate that the Becker gene is located in the same region where Duchenne has been mapped and also yield information about relative genetic distances among different DNA polymorphisms of the X chromosome.     

Mapping of locus for X-linked congenital stationary night blindness (CSNB1) proximal to DXS7.

A recombinant chromosome in a male affected with X-linked congenital stationary night blindness (CSNB1) provides new information on the location of the CSNB1 locus. A four-generation family with five males affected with X-linked CSNB was analyzed with five polymorphic markers for four X-chromosome loci spanning the region OTC (Xp21.1) to DXS255 (Xp11.22). Four of the males inherited the same X chromosome; one male inherited a chromosome that from OTC to DXS7, inclusive, was derived from the normal X chromosome of his unaffected grandfather and that from a location between DXS7 and DXS426 proximally was derived from the chromosome carrying the CSNB1 locus. This recombinant maps the CSNB1 locus in this family to a region on the short arm of the X chromosome proximal to the DXS7 locus.     

Mapping of 12 translocation breakpoints in the Xp21 region with respect to the locus for Duchenne muscular dystrophy

Over 20 females have been reported to carry reciprocal X; autosome translocations with breakpoints in Xp21 and to suffer from Duchenne muscular dystrophy (DMD). We have positioned nine of these breakpoints with respect to the Duchenne gene by mapping probes from the DMD region against a panel of somatic cell hybrids, each containing one of the translocation chromosomes from a different female patient; further information has also been obtained by in situ hybridization, including the breakpoint location in a tenth DMD patient. We have also characterized two translocation breakpoints that lie in the same chromosomal region but which are not associated with the expression of DMD. All the DMD-associated translocation breakpoints examined lie at several sites within the DMD locus and between the two non-DMD breakpoints.     

Long-range genomic map of the Duchenne muscular dystrophy (DMD) gene: Isolation and use of J66 (DXS268), a distal intragenic marker

By cloning the endpoints of a DMD-associated deletion, we have "jumped" 1100 kb from pERT87-1 (DSX164) to a new locus designated J66 (DXS268), mapping distally within the Duchenne muscular dystrophy (DMD) gene. Both J66 and JBir are mapped by field-inversion gel electrophoresis and detect abnormal SfiI fragments in DMD patients and distal DMD-associated X; autosome translocations. Our long-range map extends the physical map of the DMD gene from 800 to 2000 kb (2 Mb) and increases the mapped portion of Xp21 to approximately 8 Mb. The position of the glycerol kinase gene and the adrenal hypoplasia locus are further confined to the region between J66 and the nearest distal probe L1-4. This region spans at least 1.5 Mb. The multiallelic J66 polymorphism has immediate application in the diagnosis of DMD and generally appears to be distal to DMD mutations.     

Isolation and characterization of a highly polymorphic human locus (DXS455) in proximal Xq28

Human Xq28 is highly gene dense with over 27 loci. Because most of these genes have been mapped by linkage to polymorphic loci, only one of which (DXS52) is informative in most families, a search was conducted for new, highly polymorphic Xq28 markers. From a cosmid library constructed using a somatic cell hybrid containing human Xq27.3----qter as the sole human DNA, a human-insert cosmid (c346) was identified and found to reveal variation on Southern blot analyses with female DNA digested with any of several different restriction endonucleases. Two subclones of c346, p346.8 and p346.T, that respectively identify a multiallelic VNTR locus and a frequent two-allele TaqI polymorphism were isolated. Examination of 21 unrelated females showed heterozygosity of 76 and 57%, respectively. These two markers appeared to be in linkage equilibrium, and a combined analysis revealed heterozygosity in 91% of unrelated females. Families segregating the fragile X syndrome with key Xq28 crossovers position this locus (designated DXS455) between the proximal Xq28 locus DXS296 (VK21) and the more distal locus DXS374 (1A1), which is proximal to DXS52. DXS455 is therefore the most polymorphic locus identified in Xq28 and will be useful in the genetic analysis of this gene dense region, including the diagnosis of nearby genetic disease loci by linkage.     

Duchenne muscular dystrophy (DMD) in a female with an X/autosomal translocation: Further evidence that the DMD locus is at Xp21

An isolated case of Duchenne muscular dystrophy in a female who has a de novo t(X;5)(p21;q35) translocation is described. The similarities between this patient and four previously reported females with Duchenne muscular dystrophy are discussed. It is concluded that the locus for Duchenne muscular dystrophy is at Xp21 and, furthermore, that this site may be particularly susceptible both to chromosome breakage and exchange and to gene mutation.     

Different mosaicism frequencies for proximal and distal Duchenne muscular dystrophy (DMD) mutations indicate difference in etiology and recurrence risk.

In about 65% of the cases of Duchenne muscular dystrophy (DMD) a partial gene deletion or duplication in the dystrophin gene can be detected. These mutations are clustered at two hot spots: 30% at the hot spot in the proximal part of the gene and about 70% at a more distal hot spot. Unexpectedly we observed a higher frequency of proximal gene rearrangements among proved "germ line" mosaic cases. Of the 24 mosaic cases we are aware of, 19 (79%) have a proximal mutation, while only 5 (21%) have a distal mutation. This finding indicates that the mutations at the two hot spots in the dystrophin gene differ in origin. Independent support for the different mosaicism frequency was found by comparing the mutation spectra observed in isolated cases of DMD and familial cases of DMD. In a large two-center study of 473 patients from Brazil and the Netherlands, we detected a significant difference in the deletion distribution of isolated (proximal:distal ratio 1:3) and familial cases (ratio 1:1). We conclude from these data that proximal deletions most likely occur early in embryonic development, causing them to have a higher chance of becoming familial, while distal deletions occur later and have a higher chance of causing only isolated cases. Finally, our findings have important consequences for the calculation of recurrence-risk estimates according to the site of the deletion: a "proximal" new mutant has an increased recurrence risk of approximately 30%, and a "distal" new mutant has a decreased recurrence risk of approximately 4%.     

Assignment of a form of congenital muscular dystrophy with secondary merosin deficiency to chromosome 1q42

We have previously reported an autosomal recessive form of congenital muscular dystrophy, characterized by proximal girdle weakness, generalized muscle hypertrophy, rigidity of the spine, and contractures of the tendo Achilles, in a consanguineous family from the United Arab Emirates. Early respiratory failure resulting from severe diaphragmatic involvement was present. Intellect and the results of brain imaging were normal. Serum creatine kinase levels were grossly elevated, and muscle-biopsy samples showed dystrophic changes. The expression of the laminin-alpha2 chain of merosin was reduced on several fibers, but linkage analysis excluded the LAMA2 locus on chromosome 6q22-23. Here, we report the results of genomewide linkage analysis of this family, by use of homozygosity mapping. In all four affected children, an identical homozygous region was identified on chromosome 1q42, spanning 6-15 cM between flanking markers D1S2860 and D1S2800. We have identified a second German family with two affected children having similar clinical and histopathological features; they are consistent with linkage to the same locus. The cumulative LOD score was 3.57 (straight theta=.00) at marker D1S213. This represents a novel locus for congenital muscular dystrophy. We suggest calling this disorder "CMD1B." The expression of three functional candidate genes in the CMD1B critical region was investigated, and no detectable changes in their level of expression were observed. The secondary reduction in laminin-alpha2 chain in these families suggests that the primary genetic defect resides in a gene coding for a protein involved in basal lamina assembly.     

Studies on muscular dystrophy: a comparison by physical and chemical means of the normal human ATP-creatine transphosphorylases (creatine kinases) with those from tissues of Duchenne muscular dystrophy.

The four human Duchenne dystrophic isoenzymes (M-M, M-B, B-B, from the muscle and B-B from the brain) of ATP-creatine transphosphorylase (S. A. Kuby, H. J. Keutel, K. Okabe, H. K. Jacobs, F. Ziter, D. Gerber, and F. H. Tyler, 1977, J. Biol. Chem.252, 8382–8390) have now been compared physically and chemically with their normal human counterparts (viz., with the three isoenzymes, M-M, M-B, B-B, 2). All isoenzymes proved to be composed of two noncovalently linked polypeptide chains, by sedimentation equilibrium analyses in the presence and absence of disruptive agents. In the presence of 2-mercaptoethanol at 0.16(Γ/2), pH 7.8, the two native muscle types yielded identical values for s_20,w, concentration dependencies, and molecular weight, and similarly for the brain types (from the brain). But the human brain type proved to be slightly heavier than the muscle type (viz. 88,400 vs 85,900). All of the isoenzymes showed similar electrophoretic behavior between their several counterparts between pH 5–8, except perhaps between pH 8–10, where small differences appeared. The three native normal human isoenzymes, as well as the dystrophic human isoenzymes (M-M from the muscle and B-B from the brain) all contain 2 reactive sulfhydryl groups per mole or 1 per polypeptide chain of these two-chain proteins, which may be titrated with 5,5′-dithiobis(2-nitrobenzoic acid) (Nbs₂); and under acidic conditions, quantitative titrations with 4,4′-dithiodipyridine yield a total of 10 -SH groups per mole of each brain type and 8 -SH groups per mole of muscle type, in the case of man, dystrophic man, calf, and rabbit. The kinetics of reactions between Nbs₂ and the sulfhydryl groups of all three normal human isoenzymes and two dystrophic human isoenzymes have been measured under several sets of denaturing conditions. A comparison of their reactive calculated second-order velocity constants reveal significant differences between these three normal human isoenzymes, but the k_{second order} values for the reactions of the sulfhydryl groups of the dystrophic M-M and B-B with Nbs₂, when compared with their normal counterparts, gave identical values in the presence of 7.3 m urea or 1.8% laurylsulfate, from which it may be inferred that very similar, if not identical, environments surround these two sets of sulfhydryl groups. A comparison of the amino acid compositions of the normal human muscle type and brain type with the human dystrophic M-M and B-B (from the brain) reveal essentially identical values for the muscle types but nearly identical values for the brain types, with a few differences. Their respective tryptic peptide maps have been compared of the S-carboxy-methylated proteins (alkylated with iodo[2-¹⁴C]acetic acid at the two exposed -SH groups per mole). Thus, the muscle types, normal and dystrophic, yield identical maps, but the brain types nearly identical maps, with a few significant differences. Isolation of the tryptic tridecapeptide from the S-carboxymethylated normal human and dystrophic human dimeric muscle-type ATP-creatine transphosphorylases, labeled at the single exposed SH group per polypeptide chain with iodo[2-¹⁴C]acetate, yielded the following sequence for both proteins: ValLeuThrCys(CH₂COOH)ProSerAsnLeuGlyThr GlyLeuArg [where Cys(CH₂COOH) is S-carboxymethyl cysteine]. This sequence showed remarkable homology with a few other equivalent peptides reported to be derived from the exposed SH group of other ATP-creatine transphosphorylases. In conclusion, there does not appear to be a mutation in the structural genes for the muscle-type creatine kinases detectable by the analyses presented here. However, the brain types warrant further investigation.     

Cell growth and substrate effects on characteristics of a lysosomal enzyme (cathepsin C) in Duchenne muscular dystrophy fibroblasts

Skin fibroblasts from normal males and males suffering from Duchenne muscular dystrophy were studied in culture over a 10-week period. The lysosomal enzyme cathepsin C (dipeptidyl aminopeptidase I; EC 3.4.14.1), defined by the chloride-dependent hydrolysis of dipeptide-beta-naphthylamide (dipeptide-beta-NA) substrates at pH 5.1, was significantly lower in Duchenne cell sonicates and cell lysosomal preparations. The apparent difference in activity tended to increase with in vitro cell culture age, with the Duchenne cells being found also to grow faster and yield a greater number of cells at confluence. An analysis of all 10 cell lines as a group indicated that cathepsin C activity was related to growth rate. In addition, while analyses of cell homogenization and fractionation showed that the yield of cathepsin C was not different in Duchenne lysosomal preparations, the enzyme showed significantly lower latent activity in the Duchenne lysosomes with Gly-Phe-NA used as substrate. However, despite significant differences in specific activity compared with normal lysosomal preparations, no latency difference was observed if three other substrates were used (Gly-Arg-, Pro-Arg-, and Pro-Phe-NAs). The expression of this enzyme can thus be differentially influenced by cell growth and its latency characteristics can be influenced by the substrate used in assays.