Physical mapping distal to the DMD locus

GENOMICS

8,106-112

(1990)

Physical Mapping

Distal to the DMD Locus

D. R. LOVE, J. F. BLOOMFIELD, 5. J. KENWRICK,’ institute

of Molecular

J. R. W. YATES,*

AND

K. E. DAVIES

Medicine, lohn Radcliffe Hospital, Headington, Oxford OX3 SW, England; and *Department University of Cambridge, Tennis Court Road, Cambridge CB2 1 QP, England Received

February

19, 1990;

revised

May

of Pathology,

7. 1990

mosome spreads and therefore are likely to be greater than 2-3 Mb. These large and in some casesnon-overlapping deletions are consistent with the lack of close physical linkage between the 3’ end of the dystrophin gene and the distal loci DXS28 and DXS68 as assayed by pulsed-field gel electrophoresis (PFGE; Burmeister et al., 1988). Thus, new DNA markers are required to determine the distribution of gene sequences in this region and to complete the physical map of Xp21. We have cloned the Xp21 junction fragment of a deletion in a patient suffering from GK and DMD but not AHC. Pulsed-field gel electrophoresis studies using the distal portion of this fragment demonstrate a relatively large distance between DXS28 and the 3’ end of the dystrophin gene.

We report a new locus, designated JC- 1, which maps between the gene responsible for adrenal hypoplasia (AHC) and the gene that encodes glycerol kinase (GK) in Xp2 1.2-2 1.3. The probe identifying this locus was obtained by cloning the distal sequence of a junction fragment from a Duchenne muscular dystrophy (DMD) patient with a large deletion. Pulsed-field gel electrophoresis analysis shows that a region of at least 4 Mb separates the 3’ end of the dystrophin gene and the closest distal marker to AHC, DXS28. This region of the human genome contains few genes whose deletion results in a clinical phenotype. JC-1 is a useful probe from which to initiate strategies directed at cloning the AHC and GK loci. o 1000 Academic press, IN.

MATERIALS

INTRODUCTION

AND

METHODS

Cloning of the Junction Fragment of Patient 1477

The diseaseloci for adrenal hypoplasia (AHC), glycerol kinase (GK) deficiency, and Duchenne muscular dystrophy (DMD) have all been mapped within the Xp21 band of the human X chromosome. The order Xpter-AHC-GK-DMD-Xcen has been established by the analysis of deletions in patients suffering from various combinations of the three syndromes (Patil et al., 1985; Wieringa et al., 1985; Bartley et al., 1986; Wilcox et al., 1986; Dunger et al., 1986; Francke et al., 1987; Marlhens et al., 1987; Yates et al., 1987; Davies et al., 1988; Darras and Francke, 1988; Towbin et al, 1989; McCabe et al., 1989; Mandel et al., 1989). Studies of several patient DNAs have placed DXS68 (Ll) as the most proximal probe distal to the AHC gene, although more recent data have suggested that DXS28 (C7) may be closer to AHC than DXS68 (Bartley and Gies, 1989; Mandel et al., 1989). The order DXSG&DXS28-AHCXcen has recently been confirmed by the analysis of other patients (Towbin et al., 1990). Most of the X-chromosome deletions described above are detectable cytogenetically in extended chro-

Chromosomal DNA from a lymphoblastoid cell line of patient 1477 was digested to completion with XbaI, and 0.25 pg was ligated in 5 ~1 with 1 /rg of XbaI plus EcoRI-digested EMBLl2 DNA (Natt and Scherer, 1986). The DNAs were incubated at 14’C overnight and diluted to 500 ~1 in TMN (10 mA4 Tris-HCl, pH 7.4, 10 m&f MgClz, 100 mM NaCl), and 25 j.d of this mixture was packaged in vitro using Gigapack Gold (Stratagene) according to the manufacturer’s instructions. Approximately 4 X lo4 phages were plated to NM621, and three plaques that hybridized with the l.O-kb EcoRI-PstI fragment of the dystrophin cDNA fragment Ca38 were identified (seeFig. 1 for restriction enzyme map of this cDNA). Of the three phages detected by Ca38, one phage contained the XbaI junction fragment of patient 1477. This XbaI fragment (J-JC) was isolated from the recombinant phage DNA and cloned into pUC13 for subsequent mapping and subcloning.

1 Present address: versity of California,

DNA was prepared in agarose blocks and digested as described previously (Kenwrick et al., 1987). Samples

Howard Hughes San Francisco,

Medical Institute, U-426, California 94143-0724.

o&33-7543/90$3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Pulsed-Field Gel Electrophoresis

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PHYSICAL

MAPPING

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FIG. 1. Location of the proximal deletion endpoint and hybridization analysis of patient 1477. (A) Restriction enzyme map of cDNA Ca36 isolated from a human adult muscle library. The locations of EcoRI (E), BglII (Bg), PstI (P), and Xba (X) sites are indicated. The P&I sits corresponds to nucleotide position 5456 in the human fetal muscle transcript of the dystrophin gene (17). (B) Schematic representation of the order and sizes of the exon-containing (denoted E37-E43) genomic Hind111 fragments (denoted H27-H31) identified by the l.O-kb EcoRI-PstI fragment of Ca38. The vertical arrow indicates the relative location of the proximal deletion endpoint of patient 1477. (C) Hybridization of HindHI-digested DNA from a normal individual (lane 1) and patient 1477 (lane 2) with the l.O-kb EcoRI-P&I fragment of Ca38. Each exon-containing fragment is numbered according to B, and the dot indicates a partial digestion product. (D and E) Blots of digested DNAs from a normal male (lanes 1, 3, 6, and 7) and patient 1477 (lanes 2, 4, 6, and 8) were hybridized with the l.O-kb EcoRIPstI fragment of Ca38 (D), stripped, and rehybridized with JC-0 (E; see text and Fig. 2A for description of probe) under competition. The arrowheads in D indicate exon 42-containing fragments; these fragments are of an altered size for every digest of patient 1477 DNA, compared with those seen in a normal individual. The dots indicate partial digestion products. The dark arrowheads indicate the XbaI junction fragment (J-JC; see Fig. 2A) of patient 1477.

included sperm and a 48,XXXX lymphoblastoid cell line (GM1416). Electrophoresis was carried out in 0.70.8% agarose using an OFAGE and a hexagonal electrode array apparatus from Pharmacia LKB and also a CHEF apparatus built to the design of Chu (Chu et aZ., 1986; Vollrath and Davis, 1987). The gel running conditions are given in the figure legends. Molecular weight markers were X concatemers (Fig. 3), together with chromosomes of Saccharomyces cerevisiae, Candida albicans, and Schizosaccharomyces pombe (FMC Corp.; Fig. 4).

Southern

Blotting

and Hybridization

Conventional and pulsed-field agarose gels were blotted to nylon membrane (Hybond N, Amersham) and hybridized as described previously (Kenwrick et al., 1987). Purified DNA fragments were labeled by the random priming method of Feinberg and Vogelstein (1983). The probe JC-0 was hybridized under competition by the method described by Sealey et al. (1985); the probe and competitor DNAs were incubated at 65°C for 30

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FIG. 2. Analysis of XboI junction fragment of patient 1477. (A) Restriction enzyme map of XboI junction fragment; sites are indicated as described in Fig. 1, together with those for Hind11 (H2), HMIII (H3), and KpnI (K). The filled and hatched boxes indicate exon 42containing and repeated sequences, respectively. These sequences were identified by hybridization with Ca38 and labeled human chromosomal DNA (results not shown). (B and C) Hybridization of digested chromosomal DNA from patients suffering complex phenotypes, with JC-1. The locations of size markers are indicated in kilobase pairs. Lanes 1,8, 6, and 7 of C contain DNA from a normal individual, and lanes 2, 4, 6, and 8 contain 2468 DNA. (D) Physical map of Xp21 indicating relative locations of loci (5); the top-most horizontal line indicates intervals of 1 Mb, with the region bounded by DXS28 and DXS268 representing at least 4 Mb (see text). The extent and location of deletions in several patient DNAs ara indicated by open boxes (34); ses also (19). The dashed lines indicate uncertainty regarding the extant of a deletion; the 3’ deletion endpoint in patient 1290 is further distal than that in patient 1477 in view of the clinical phenotypes manifested by these patients, The clinical phenotypes are indicati DMD (Duchenne muscular dystrophy); GK (glycerol kinaee deficiency); MB (mental retardation); and AHC (adrenal hypoplasia).

PHYSICAL

MAPPING

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FIG. 3. PFGE mapping of Xp21 probes to Sfi I-digested DNA. Sperm DNAs from several normal individuals were digested to completion with Sfi I and electrophoresed at 400 V for 20 h with a 45-s pulse time. The DNAs were blotted and hybridized sequentially with dystrophin gene probes DXS270 (JBir), DXS268 (J66), 1.2-kb HindI fragment of Cf115 (cDNA containing the 3’ end of the dystrophin gene (14)), and finally with JC-0. The schematic diagram represents a physical map of the region that encompasses the probes used. The arrow indicates the direction of transcription of the dystrophin gene. The filled and open circles denote Sfi I sites that are digested to completion and partially digested, respectively (lengths of fragments deduced from autoradiographs presented and order of fragments taken from den Dunnen et al. (10)). The dashed line indicates the lack of physical linkage between Sfi I fragments detected by JC-0 and the 3’ end of the dystrophin gene.

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RESULTS Patient 1477 (JC) suffers from DMD and GK deficiency but not AHC and has been reported previously (Davies et al., 1988). The characterization of his deleted X chromosome using cDNA probes derived from the dystrophin gene (Forrest et al., 1987, 1988) demonstrates that 1477 is deleted for all exons 3’ of exon 42, which lies near the middle of the gene. The position of the proximal breakpoint relative to the order of dystrophin exons (Koenig et al., 1989) is illustrated in Fig. 1. This breakpoint can be detected as an altered size fragment on Southern blots of digested 1477 DNA probed with a cDNA containing exon 42 (Fig. 1D). We chose to clone the high-molecular-weight junction fragment identified by XbuI digestion, as its length was thought to offer a greater likelihood of isolating singlecopy DNA sequence from the distal side of the breakpoint. A restriction enzyme map of the cloned XbaI fragment is shown in Fig. 2A. Exon 42 is situated at the centromeric end of this fragment (results not shown). Therefore, sequences distal to the PstI site nearest exon

42 should be derived from the distal side of the breakpoint because an altered size PstI fragment is detected with Ca38 (Fig. 1D). Indeed, an X&I-PstI fragment derived from the junction fragment (JC-0; Fig. 2A) clearly identifies the novel exon 42-containing XbaI fragment of patient 1477 (Fig. 1E). Two genomic KpnI fragments hybridize with JC-0 because the probe has a KpnI site within it (Fig. 2A). Verification that JC-0 was indeed derived from the distal region of the junction fragment was obtained by hybridizing the single-copy XbaI-KpnI fragment (JC1) to DNA from other patients with deletions in the GK-AHC region (Figs. 2B and 2C). JC-1 is deleted in patients with extensive Xp21 deletions encompassing the GK and AHC loci (patients 1290 and 346) but is present, as expected, in patient 1477 from whom JC1 was isolated. JC-1 is detected in patient 1400, who has a 6000-kb deletion of the DMD locus but does not suffer from glycerol kinase deficiency (Wilcox et al., 1986). JC-1 is also present in patient 2408 (Fig. 2C), who suffers from adrenal hypoplasia but not glycerol kinase deficiency (Yates et al., 1987). No altered fragments are detected by JC-1 after hybridization to several different restriction enzyme digests of 2408 DNA (Fig. 2C). These data localize JC-1 between the GK and AHC loci and indicate that the distal deletion end-

110

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FIG. 4. PFGE mapping of Xp21 probes to EssHII-digested DNA. Digested sperm DNAs were electrophoresed at 55 V for 5 days with a ZO-min pulse time. The DNAs were hybridized initially with DXS268 (A) and DXS68 (B), followed by JC-0. The higher molacular weight fragments detected by DXS268 and DXS68 represent partial digestion products. DNA isolated from a 44XXXX cell line (GM1416) was digested partially (time course) with BssHII and electrophoresed at 45 V for 140 h with a pulse time that was ramped linearly from 90 to 60 min. The DNAs were blotted and probed sequentially with JC-1, DXS68, and DXS28 (C). The filled arrow heads indicate fragments of approximately 3.5 and 4.2 Mb (JC-1) and 3.8 Mb (DXSSS).

point of patient 1477 is not within 12 kb of the proximal deletion endpoint in patient 2408. Previous pulsed-field gel electrophoresis analyses have failed to link the 3’ end of the dystrophin gene with the loci DXS28 and DXS68, distal to AHC (Burmeister et al., 1988). We decided therefore to investigate whether physical linkage of JC-0 and JC-1 to the dystrophin gene and to the more distal loci could be established using PFGE. We chose the restriction enzymes Sfi I and BssHII for this analysis because digestion with these enzymes results in large fragments that have been previously documented for several markers in Xp21 (Burmeister and Lehrach, 1986; Kenwrick et al., 1987; Burmeister et al., 1988, den Dunnen et al., 1989). Figure 3 shows that JC-0 and probes from the

ET

AL.

3’ end of the dystrophin gene identify different SfiI fragments; the terminal 650-kb Sfi I fragment of the dystrophin gene identified by the 1.2-kb Hind11 fragment of Cf115 also hybridizes with the 3’ end fragment of the dystrophin transcript (results not shown). JC-0 hybridizes to two SjiI fragments of 350 and 80 kb, compared with the 680- and 310-kb SfiI fragments which have been reported to be identified by DXS28 and DXS68, respectively (van Ommen et al., 1986); the 310-kb SjiI fragment identified by DXS68 was confirmed in this study but results are not shown. The probe JC-0 does not contain an Sfi I site; therefore, the two genomic Sfi I fragments are likely to be due to partial digestion. The genomic analysis was extended with BssHII because digestion with this enzyme generates larger fragments for JC-0 and JC-1 compared with the use of Sji I (Fig. 4). DXS268, JC-0, and DXS68 identify independent BssHII fragments in complete digests of sperm DNA. The BssHII fragments identified by DXS268 also hybridize with the 3’ end of the dystrophin transcript, and hence represent the telomeric end of the dystrophin gene, and DXS28 identifies an 840kb BssHII fragment which confirms previously published data (results not shown; Burmeister et aZ., 1988). JC-1 hybridizes with fragments of approximately 3.5 and 4.2 Mb in partial BssHII digests of DNA from a 48,XXXX cell line (GM1416) that are not detected by either DXS28 or DXS68. The large BssHII fragments that hybridized with JC-1 were subsequently shown not to hybridize to DXS268 (results not shown).

DISCUSSION We have isolated a new DNA sequence that maps between GK and AHC on the human X chromosome. Physical mapping of this locus has failed to link it physically either with the 3’ end of the dystrophin gene or with DXS28, which lies distal to AHC. The genomic region between DXS28 and the 3’ end of the dystrophin gene therefore must be greater than 4.2 Mb, which is the size of the larger partial BssHII fragment detected hy JC-1. The precise positions of AHC and GK within this region have yet to be determined. JC-1 detects large pulsed-field gel fragments with Sfi I and BssHII, which suggests a paucity of HTF islands (Bird, 1986) in the DMD-GK-AHC region. Thus, unlike the fragile X region of the X chromosome in XqZir-qter (Patterson et al., 1988,1989), the Xp21 region may be relatively AT-rich, which would suggest that it contains few genes. This is compatible with the fact that the deletion of portions of Xp21.3-Xp21.1 results in AHC, GK, DMD, and perhaps Aland eye disease (Mandel et uL, 1989; McCabe et al., 1989) but no other easily recognizable clinical phenotype.

PHYSICAL

MAPPING

JC-1 should prove useful in analyzing the GK-AHC region by genetic and physical mapping studies of DNA from normal individuals and patients with clinical phenotypes relevant to this region of the X chromosome. The isolation of cosmids and YACs that contain JC-1 should enable further probes to be isolated, both proximal and distal to JC-1. These probes could be used to identify expressed sequences,although the distances involved may militate against their use in identifying the GK and AHC genes. A more amenable strategy to determine the relative position of the GK gene would be to hybridize JC-1 with DNAs from patients manifesting GK only, but with cytogenetically undetectable rearrangements. This strategy would determine whether JC-1 is frequently deleted in such patients and also its proximity to the GK gene itself. ACKNOWLEDGMENTS We thank Martyn Bell for his advice with PFGE and construction of the CHEF apparatus, Michael Murphy for help in isolating J-JC, and Helen Blaber for help in the preparation of the manuscript. We are grateful to the Medical Research Council of Great Britain, the Muscular Dystrophy Group of Great Britain, and the Muscular Dystrophy Association, USA, for financial support. REFERENCES 1. BARTLEY, J., AND GIES, C. (1989). A Xp21 deletion assigns locus DXS28 (C7) proximal to DXS68 (L1.4) and DXS67 (B24) and has the proximal breakpoint in the intron 3’ to the first exon of DMD-8: HGMlO. Cytogenet. Cell Genet. 61: 958-959. 2. BARTLEY, J. A., PATIL, S., DAVENPORT, S., GOLDSTEIN, D., AND PICKENS, J. (1986). Duchenne muscular dystrophy, glycerol kinase deficiency, and adrenal insufficiency associated with Xp21 interstitial deletion. J. Pediatr. 108: 189-192. 3. BIRD, A. P. (1986). CpG-rich islands and the function of DNA methylation. Nature (London) 321: 209-213. 4. BURMEISTER, M., AND LEHRACH, H. (1986). Long-range restriction map around the Duchenne muscular dystrophy gene. Nature

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DISTAL

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TO DMD

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vector Nucleic

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12, a new lambda I, Barn

HI,

Sst I

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30.

31.

32.

33.

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