Mapping DNA Sequences in a Human X-Chromosome ... - Europe PMC

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K. HAAPALA,3 G. BATES,1 H. F. WILLARD,4 P. PEARSON,5. AND K. E. ... indicates that in at least three cases the breakpoint may be different (Y. Boyd ... Probes C7 and M2C were a gift from J.-L. Mandel (Strasbourg); probe pXBRI was a gift.
Am J Hum Genet 37:451-462, 1985

Mapping DNA Sequences in a Human X-Chromosome Deletion Which Extends across the Region of the Duchenne Muscular Dystrophy Mutation C. INGLE,' R. WILLIAMSON,' A. DE LA CHAPELLE,2 R. R. HERVA, K. HAAPALA,3 G. BATES,1 H. F. WILLARD,4 P. PEARSON,5 AND K. E. DAVIES 6

SUMMARY

A somatic cell hybrid has been constructed and characterized using fibroblasts from a phenotypically normal woman who possesses an X chromosome with an interstitial deletion of the short arm. Highresolution banding indicates that the deleted segment is either Xp22. 13-p 1 1.4 or Xp22. II-pI 1.23. Southern blot hybridization to previously mapped DNA sequences confirms that the missing segment of the X chromosome is a deletion and not an interstitial translocation and supports the cytogenetic interpretation that the deletion extends proximal of Xpl 1.3 and therefore probably comprises Xp22. 11p11.23. Three further DNA sequences have been localized to the region of the deleted segment. The following order has been assigned to the seven probes used: Xpter-RC8-pXUT22-(OAIC7,M2C)-L1.28-RD6Xcen.

Received August 21, 1984; revised November 9, 1984. This work was supported by grants from the Muscular Dystrophy Group (U.K.), the Muscular Dystrophy Association (U.S.A.), the U.K. Medical Research Council, the Sigrid Juselius Foundation, the Medical Research Council of Canada, and the Academy of Finland. Part of this study was carried out at the Folkhalsan Institute of Genetics, Finland. ' Department of Biochemistry, St. Mary's Hospital Medical School, University of London, London, W2 IPG, England. 2 The Department of Medical Genetics, University of Helsinki, Finland. 3Department of Pathology, University of Oulu, Oulu, Finland. 4 Department of Medical Genetics, University of Toronto, Canada. 5Department of Human Genetics, University of Leiden, Holland. 6 Present address: Molecular Genetics Group, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford, OX3 9DU, England. Request reprints from K. E. D. C 1985 by the American Society of Human Genetics. All rights reserved. 0002-9297/85/3703-0002$02.00

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INGLE ET AL. INTRODUCTION

Three main physical methods are used to localize DNA sequences along a chromosome: in situ hybridization to metaphase spreads, hybridization dosage studies using cell lines in which one of the pair of chromosomes is deleted for a known region, and hybridization to DNA from somatic cell hybrids containing portions of a human chromosome. In situ hybridization can only locate probes to within the precision of the cytological identification of the chromosome region and the spread of the radioactive grains [1]. It is a time-consuming method and unsuitable for screening a large number of probes. Dosage analysis using deletions [2] can be used for the location of probes to regions of the chromosome, but the interpretation of such data can be equivocal because signal intensity can vary for reasons other than gene dosage. The most rapid method of localizing recombinant DNA sequences to a chromosomal region is by the use of somatic cell hybrids. These can be constructed to include only portions of a human chromosome by using, as the human partner in the fusion cell, lines with balanced translocations, deletions, or rearrangements [2, 3] or by using fragments generated by irradiating donor cells [4]. We have constructed a human/rodent cell hybrid containing an X chromosome with a deletion of the Xp21 band for use in the rapid identification of DNA sequences located in this region. The Xp2l region of the human chromosome complement is of particular interest in efforts to identify the basic defect involved in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). Although both DMD and BMD have been shown by linkage analysis to map to the general region of Xp2l [5, 6], the exact location is not known. A number of female cases of apparent Duchenne or Becker phenotype have been studied. In some of these, an X-autosome translocation involving Xp2l has occurred. High-resolution analysis of prometaphase chromosomes indicates that in at least three cases the breakpoint may be different (Y. Boyd and V. J. Buckle, personal communication, 1985). In addition, new mutations occur at high frequency and the phenotype is variable in both boys and the rare female cases. These observations may indicate that the disease is heterogeneous and that there is more than one mutation in the Xp21 region that gives rise to the DMD or BMD phenotype. One approach to the identification of the basic defect in these disorders is to identify functional sequences located in and adjacent to Xp2l [2, 3]. If, for example, the mutation is in a gene expressed in muscle and a random distribution of expressed sequences throughout the genome is assumed, there should be in the order of 400 sequences located on the X chromosome that are expressed in muscle. (This assumes that approximately 8,000 genes are expressed in muscle and that the X chromosome is 5% of the total haploid genome content.) Since the region Xp2l comprises 10% of the total X chromosome, only 40 of these will be located in this region. Thus, a rapid method of localizing DNA sequences to the region of the Xp2l band would be valuable in the search for the basic defect and will also contribute to an understanding of the distribution of functional sequences in this region of the human X chromosome.

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MATERIALS AND METHODS

Hybrid Cell Construction Somatic cell hybrids were constructed from an established fibroblast cell line of a phenotypically normal woman who has an X chromosome with a deletion in the region of Xp2l [7] and a tk- subtetraploid Chinese hamster lung cell line: a23 [8]. Fusion was performed with PEG 6000, and hybrids were selected in Hamm's F-12 HAT-selective medium and ouabain [9]. Other hybrid cell lines used were a gift from Prof. H.-H. Ropers. DNA Probes The following DNA probes were used: RC8, 77A (a DNA sequence detecting the same polymorphism as RC8), L1.28, S9, 52A, HPRT, and DX13. Full details of probes and hybridization conditions, including the stringency to which Southern blots were washed after hybridization with each probe, are to be found in previous studies [10, 11]. Probes C7 and M2C were a gift from J.-L. Mandel (Strasbourg); probe pXBRI was a gift from B. A. Hamkalo (San Diego, Calif.).

DNA Extraction DNA was extracted from approximately 107 hybrid cells as follows: after washing with phosphate-buffered saline containing 0.2% EDTA, cells were treated in the tissueculture flasks with proteinase K (2.5 mg in 10 ml STE, 0.5% SDS per 175 cm2 Nunclon flask). After overnight incubation at 550C, the lysate was extracted twice with phenol, twice with chloroform:isoamyl alcohol 24:1, precipitated with ethanol, and dissolved in Tris/EDTA buffer. This rapid method provided DNA suitable for digestion with restriction enzymes. Restriction Enzyme Analysis Ten ,ug of DNA from each of the hybrid cell lines was digested with restriction enzymes (B.R.L., Cambridge, England) according to the manufacturer's instructions, separated by electrophoresis on 0.8% agarose cells, and transferred to Schleider and Schull BAS5 nitrocellulose or Zetapore filters [12]. DNA probes were labeled with [32P]dCTP to a specific activity of 5 x 107 dpm/,ug DNA and hybridized to the Southern blots as described [11]. Autoradiography was performed using either Kodak or Fuji (preflashed) medical X-ray film exposed for 2-14 days at - 70°C. In Situ Hybridization In situ hybridization was carried out as described by Hartley et al. [13]. The specific gravity of the probe was 2 x 108 dpm/,ug DNA. Ilford K2 emulsion was used and autoradiographs allowed to develop for 19 days. For the scoring of the grain distribution over the whole chromosome spread, the human karyotype was divided into 110 equal sections; for finer analysis of grain distribution, the short arm of the X chromosome was divided into five sections. RESULTS

A deleted X chromosome was found to be segregating in a family investigated on account of a mentally retarded female (111-26 in published pedigree [7]). An identical deleted X chromosome also occurred in the mother (11-7) and the sister (111-27) who are phenotypically normal; therefore the proband's mental retardation could not be ascribed to the chromosomal deletion.

454 High-Resolution Banding

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Cultures of lymphocytes from the peripheral blood of all three family members carrying the deletion X chromosome were made in an attempt to localize the interstitial breakpoints more precisely by cytogenetic methods. The deletion was identical in all three individuals (mother, 11-7; proband, 111-26; sister, 111-27). Figure 1 shows both X chromosomes (G-banded) in three cells from subject 11-7. The darkly staining band Xp2l is absent in the deleted chromosome. Measurements indicate that parts of the weakly staining adjacent region are also missing. The deleted short arm contains four darkly staining bands of approximately equal intensity (fig. 1). These are four of the five narrower dark bands present on the normal Xp (i.e., bands 22.32, 22.2, 22.12, 11.3, and 11.22 [fig. 2]). As is often the case, unequivocal identification of such minor bands is not possible. We conclude, therefore, that the missing minor dark band is either 22.12 or 11.3. These results allow two possible interpretations of the extent of the deletion (fig. 2). The more distal location for the deletion (large arrows) implies the structure Xpter-p22. 12: :pl 1 .4-qter, the deleted portion being Xp22.13-pll.4. The more proximal location for the deletion (small arrows) implies the structure Xpter-p22.11::plI.23-qter, the deleted portion being Xp22.1 1-pl 1.23. With present-day cytogenetic methodology, it is not possible to make a firm statement in favor of either interpretation. Fibroblasts from the phenotypically normal sister with the deleted X chromosome (111-27; here labeled PS) were used to prepare Chinese hamsterhuman hybrid cell lines. Cloned cell lines were isolated containing each of the X chromosomes separately in a Chinese hamster background. These lines are designated Sin. Identification of the two homologs of a chromosome pair may be accomplished using heterozygosity for restriction fragment length polymorphisms (RFLP). DNA from the cell line (111-27 in the pedigree here designated PS) used in the hybrid fusion and DNA from her phenotypically and cytogenetically normal sister (111-24, here designated MJ) was tested for heterozygosity with a series of polymorphic DNA probes known to be located at sites along the full length of the X chromosome: RC8, Ll.28, S9, 52A, HPRT, DX13 [3, 12]. Although no heterozygosity was detected, the intensity of the hybridization signal on Southern blots indicated that probe Ll.28, which is known to be localized at Xpl 1. 1-pI 1.3 [3], was present in only a single copy in the cell line carrying the deletion (PS) but in two copies in the cell line carrying the normal X (MJ). These data also demonstrate that the deleted portion of the X chromosome is not translocated to another region of the genome. A series of random cloned cell lines were isolated from a somatic cell fusion of the human fibroblast line (PS) with a tk- Chinese hamster lung cell line (a23). Selection in HAT medium was therefore for the human tk- gene on chromosome 17, and both active and inactive X chromosome would be expected among the hybrids selected. DNA from the selected hybrids was digested with TaqI and hybridized to two DNA probes. Probe 52A, located at Xq27 or Xq28 (table 1), gave a positive DNA hybridization signal with all cell hybrids carrying

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FIG. 1.-Cutout X chromosomes from three G-banded lymphocyte mitoses of subject 11-7 in the previously published pedigree [7] showing normal (left) and deleted (right) X chromosomes.

an X chromosome. Probe Li.28 located at Xqll.1-Xpll.3 gave a positive signal with cell hybrids carrying a complete X chromosome and a negative signal with cell hybrids carrying the deleted X chromosome (table 1). Of 12 somatic cell hybrids examined, five gave a positive signal with probe 52A, and of these, two gave no signal with Li.28. Two cell hybrids were chosen for further study; one, designated Sin 176(delX), carries the deleted X chromosome, and one, designated Sin 22(X), which shows an equal intensity of hybridization with Li.28 and 52A indicating a single copy of each gene, is presumed to carry only the normal X chromosome. 2 2 33 22 32

22 31 22 2 22.13 22 12 22 1 1 21 3 21 2 21

11

4

1. 3 11 23 11 .22

11 21 112

12

13 1 13 2 13 3 21. 1 21 2 21 31 21 .32 21 33 22 .1 22 2 22 3 23 24

25 26 1 266 2 26. 3

27.1 27. 2 27 3 28

FIG. 2.-Prometaphase G-banding pattern of normal X chromosome (approximately 850-band stage). Large arrows indicate extent of the more distal, and small arrows, extent of the more proximal alternative interpretation of the deletion.

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TABLE 1 ASSIGNMENTS OF X CHROMOSOME PROBES TRANSLOCATION HYBRIDS ASSIGNMENT IN SITU

CLONE

77A ........ pXUT22 .... 0Al .........

Xp22.3-p21 Xp22-p21

Xp2I -Xqter

Xp I .3-Xqter

Xp I I-Xqter

Sin 176 (delX)

Sin 22 (X)

...

...

...

+ +

+ + +

...-

...

...

-

........

...

...

...

-

C7 ..........

...

-...

...

-

+

L1.28

...

+

-

-

+

+ + ...

... ...

+ + +

+

M2C

.......

RD6....

pXBRI ..... 52A ........

Centromere Xq27 or Xq28

...

...

... ...

-

+

+

+

Karyotyping of the Hybrid Sin 176(delX) Seventeen trypsin-banded metaphases of cell line Sin 176(de IX) were stained with Giemsa, and 16 clearly showed the presence of the deleted X chromosome. The banding pattern, showing the complete absence of Xp21, is in agreement with the report of Herva et al. [7] and the high-resolution banding study reported here. The normal X chromosome was not observed in any spread. Other human chromosomes identified in Sin 176(delX) were 2, 3, 4, 5, 6, 8, 11, 13, 16, 17, 19, and 20. These chromosomes were present in the majority of spreads examined.

Analysis with DNA Probes The extent of the deletion was mapped using several DNA probes whose locations were already known. DNA samples from hybrid cell lines Sin 176(de IX) and Sin 22(X), the fibroblast parent of the hybrid cell lines (PS), and total genomic DNA (G) were digested with the restriction enzyme Taql. After Southern blotting, they were hybridized to DNA probes 77A, Li.28, 52A, and RD6 [3, 10]. The results obtained are shown in figure 3 and summarized with other localization data in table 1. PS shows one band at the L1.28 locus, and MJ is heterozygous for the locus with two bands. This TaqI polymorphism has been reported [11]. The lower intensity of the 1.7 kilobases (kb) in RD6 is a blotting artifact since it is not reproducible at this intensity. All the probes studied with the exception of Li.28 give signals with DNA from both hybrid cell lines; therefore, all the clones with the exception of LI.28 lie outside the deletion. Clones LI.28 and RD6 have both been localized to the region between Xpl 1.3 and Xpl 1 [3], and since Li.28 but not RD6 is within the deletion, this clearly defines the proximal end of the deletion as being located between Xpl 1.3 and Xpl 1. Therefore, the order of these probes is: Xpter . . . L1 .28 . . .RD6 . . . centromere. Further information with regard to the proximal end of the deletion was obtained using a clone designated pXBR1. This probe contains a BamHI tan-

457

DUCHENNE MUSCULAR DYSTROPHY C

SIN SIN

SIN SIN

PV MUI 17

MA PR IIRi

797

-105 -88

22

kb

-70a -45 -38 -3*0 -23 -1 7 -1 2

DNAprobe

RD6

L.28

SIN SIN 176 22 kb

-68

-31

-1.6

DNA probe Ila

52a

FIG. 3.-Autoradiographs of the four X-specific chromosome probes of known location (table 1) hybridized to TaqI-digested DNA from Sin 176(delX); Sin 22(X); PS original fibroblast cell line (X, delX); MJ, fibroblasts from sib to PS (XX); G, unrelated genomic DNA (XX). Ten pig of DNA (G) and the hybrid line was loaded. Five ,±g of MJ and PS were loaded.

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SIN SiN Xp G PS 176 22 113 kb

,-. .:. . . ':. .....

I

::. . j . f

FIG. 4.-Autoradiograph of probe pXBRl to TaqI-digested DNA from Sin 176(delX), Sin 22(X), PS, original fibroblast cell line (X, delX) G, unrelated genomic DNA (XX), and Xpl 1.3 hybrid containing only the Xpl I .3-Xqter region of the human X chromosome.

dem repeat sequence known to occur predominantly in the centromeric region of the human X chromosome [14, 15]. A complex band pattern is obtained using DNA digested with the restriction enzyme TaqI (fig. 4G) consisting of a series of major bands at 3.3, 3.0, 2.0, 1.8, and 1.53 kb. All these bands are visible when this probe is hybridized to TaqI-digested DNA from a cell hybrid containing only Xpl 1 .3-Xqter (fig. 4, Xpl 1.3). It is therefore concluded that the lowrepeat DNA sequences hybridizing to this probe do not extend distally beyond XpI 1.3. Hybridization of pXBRI with TaqI-digested DNA from Sin 176(de IX) shows the complete absence of the two bands at 2.0 and 3.0 kb (fig. 4). All other bands show the same relative intensity of hybridization signal as the control Sin 22(X). This indicates that either a small part of the repetitive sequence lies within the deletion or that these correspond to bands from different human autosomes present in the two hybrid cell lines. The data obtained with the DNA probe pXBR1 combined with that obtained using the DNA probe RD6 are consistent with the cytogenetic interpretation that the deletion covers the region Xp22.1-Xpl1.23, indicated by the small arrows in figure 2.

Assignment of Probes of Unknown Location More information about the distal limit of the deletion has been obtained from in situ hybridization data using a random DNA probe designated pXUT22 which gives a positive hybridization signal with Sin 176(delX) (fig. 6). The grains are centered around Xp22 (fig. 5), indicating that this probe is located at Xp22, proximal to RC8, which, under the same conditions of hybridization,

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q

x FIG. 5.-Diagrammatic representation of the distribution of grains over a normal human X chromosome after hybridization with 3-H-pXUT22. Composite of 66 metaphase spreads.

gives grains centered at Xp22.3 [13]. This is consistent with the previous assignment of pXUT22 distal to the Xp2l breakpoint in a DMD female patient [16]. The deletion therefore, although including all of the Xp2l band, does not extend far into Xp22.1. These data provide additional support for the more proximal location of the deletion (small arrows in fig. 2). The somatic cell hybrid Sin 176(delX) has been used to screen a number of X-chromosome probes, and three further probes have been assigned to the region of the deletion (fig. 6). C7 and M2C are two cDNA probes, each showing a single hybridization band with control human DNA digested with EcoRI. C7 shows a band at 5.0 kb, and M2C shows a band at 2.8 kb. Clone C7 does not give a signal when hybridized to DNA from the somatic cell hybrid containing only human Xp2l-Xqter. It is, therefore, located distal to the Xp2l breakpoint of the fragment in this somatic cell hybrid, but proximal to the location of the DNA probe pXUT22 and the proposed distal termination of the deletion in Sin 176(delX) at Xp22.11. The third probe, OA1, is a genomic recombinant [17] hybridizing to fragments from normal genomic DNA at 3.6 kb and 2.2 kb after digestion with TaqI. No signal was seen with Sin 176(delX) (fig. 6) or with a cell hybrid containing only Xp21-Xqter (results not shown). This probe therefore is located in the same region as the DNA probes C7 and M2C, at Xp2l. DISCUSSION

The hybrid cell line described here contains a human X chromosome that is deleted for all of the band Xp2l and a significant amount of Xpl 1. The deletion

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460

SIN 22 I(b

kb

U

11 I I *E

DNA probe M2C g

G 6

SIN SIN 22 716kb

C7 G MJ

SIN SIN

PS116 22 kb

.2~~~~~~~.

DNA probe

DAl

pXUT22

FIG. 6.-Autoradiographs of four X-specific chromosome probes of unknown location hybridized to EcoRI-digested DNA from Sin 176(delX); Sin 22(X); PS original fibroblast cell line (X, deiX); MJ, fibroblasts from sib to PS (XX) 0, unrelated genomic DNA (XX).

DUCHENNE MUSCULAR DYSTROPHY 461 has been characterized using cloned DNA probes that have been localized previously using a somatic cell hybrid panel [3] or by in situ hybridization [13]. Dosage studies using L1.28 showed that only one copy of this probe was present in the original female fibroblast cell line, showing that the deletion present in one of the X chromosomes was not translocated elsewhere in the genome. Significantly, this cell line hybridized to one probe localized to Xpl 1Xpl 1.3 (RD6) but not with a second probe (L 1.28) also assigned to this region [3]. The end of the deletion must, therefore, lie somewhere within Xpl 1.3. This result was confirmed by the presence of the hybrid of the major hybridization bands characteristic of the BamHI tandem repeat, pXBR1, known to be predominantly located in the centromeric region of the human X chromosome [14, 15]. The exact breakpoints defining the deletion could not be determined unequivocally by high-resolution banding. It is well known that minor bands resemble each other too much to be positively identified with certainty. Thus, the proximal breakpoint was judged to be either in Xpl 1.23, which is in accord with the above interpretation. Alternatively, the breakpoint is more distal, that is, in Xpll.4. The distal limit of the deletion was harder to define because very few DNA sequences have been precisely mapped in this region. However, the hybrid line did show a signal with probe pXUT-22 localized in the region of the interface between bands Xp21 and p22 by in situ hybridization studies. Furthermore, probe C7 believed to be distal to Xp21 was absent from the deleted X chromosome. These data are in full agreement with the cytogenetic localization of the distal breakpoint in Xp22. 1. If the breakpoint, Xp22. 1, suggested by the data presented above is correct, then probes C7 is localized to the narrow subband Xp22. 11. All these data taken together indicate the following order: C7 Xpter - pXUT-22 - M2C - L1.28 - RD6 - Xcen 0A1

This hybrid cell line is now being used to locate cDNA sequences to the band Xp2l (approximately 9,000 kb of DNA) on the human X chromosome. This is of great interest since it has been shown that the mutations responsible for both Duchenne and Becker muscular dystrophies are located within this region [11]. If the gene sequences at Xp2l that are expressed in muscle are not changed in level of expression in DMD and BMD patients, then chromosome walking techniques would need to be used to identify the mutation. These sequences will also be useful for the characterization of the translocation breakpoints found in females with DMD and their effect on the expression of genes in this region [16]. Any cDNA sequences localized in this deletion will provide new markers for carrier detection and antenatal diagnosis in affected families even if the sequences themselves are not altered in DMD or BMD patients [18, 19]. ACKNOWLEDGMENT We are very grateful to Dr. Ben Carritt (Galton Laboratories, London) for help in the hybrid construction.

462 1. 2.

3.

4.

5. 6.

7. 8.

9. 10. 11. 12.

13. 14.

15. 16.

17. 18. 19.

INGLE ET AL. REFERENCES HARPER ME, SAUNDERS GF: Localization of single copy DNA sequences on Gbanded human chromosomes by in situ hybridisation. Chromosoma 83:431-439, 1983 KUNKEL L, TANTRAVAHI U, EISENHARD M, LATT S: Regional localisation on the human X of DNA segments cloned from flow sorted chromosomes. Nucleic Acids Res 10: 1557-1578, 1982 WIEACKER P. DAVIES KE, COOKE HJ, ET AL.: Toward a complete linkage map of the human X chromosome: regional assignment of 17 cloned single-copy DNA sequences employing a panel of somatic cell hybrids. Am J Hum Genet 36:265-276, 1984 Goss SJ, HARRIS H: Gene transfer by means of cell fusion I statistical mapping of the human X chromosome by analysis of radiation induced gene segregation. J Cell Sci 25:17-38, 1983 KINGSTON HM, HARPER PS, PEARSON PL, DAVIES KE, WILLIAMSON R, PAGE D: Localisation of gene for Becker muscular dystrophy. Lancet ii: 1200, 1983 O'BRIEN T, HARPER PS, DAVIES KE, MURRAY JM, SARFARAZI M, WILLIAMSON R: Absence of genetic heterogeneity in Duchenne muscular dystrophy shown by a linkage study using two cloned DNA sequences. J Med Genet 20:249-251, 1983 HERVA R, KALUZEWSKI B, DE LA CHAPELLE A: Inherited interstitial del (Xp) with minimal clinical consequences: with a note on the location of genes controlling phenotypic features. Am J Med Genet 3:43-58, 1979 WESTERVELD A, VISSER RPL, MEERA KHAN P, BOOTSMA D: Loss of human genetic markers in man-Chinese hamster somatic cell hybrids. Nature [New Biol] 234:2024, 1971 CARRITT B, POVEY S: Regional assignments of the loci AK3. ACONS and ASS on human chromosome 9. Cytogenet Cell Genet 23:171-181, 1979 DRAYNA D, DAVIES KE, HARTLEY D, ET AL.: Genetic mapping of the human X chromosome using restriction fragment length polymorphisms. Proc Natl Acad Sci USA 81:2836-2839, 1984 DAVIES KE, PEARSON PL, HARPER PS, ET AL.: Linkage analysis of two cloned DNA sequences flanking the Duchenne muscular dystrophy locus on the short arm of the human X chromosome. Nucleic Acids Res 11:2303-2312, 1983 SOUTHERN EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503-517, 1975 HARTLEY DA, DAVIES KE, DRAYNA D, WHITE RL, WILLIAMSON R: A cytological map of the human X chromosome-evidence for non-random recombination. Nucleic Acids Res 12:5277-5285, 1984 YANG TP, HANSEN SK, OISHI KK, RYDER OA, HAMKALO BA: Characterization of a cloned repetitive DNA sequence concentrated on the human X chromosome. Proc Natl Acad Sci USA 79:6593-6597, 1982 WILLARD HF, SMITH KD, SUTHERLAND J: Isolation and characterisation of a major tandem repeat family from the human X chromosome. Nucleic Acid Res 11:20172033, 1983 WORTON RG, DUFF C, SYLVESTER J, SCHMICKLE RD, WILLARD HF: Duchenne muscular dystrophy associated with translocation of the dmd gene next to ribosomal RNA genes. Science 224:1447-1449, 1984 BENHAM F, HODGKINSON S, DAVIES KE: A GAPDH pseudogene on the human X chromosome defines a multigene family. EMBO J 3:2635-2640, 1984 HARPER PS, O'BRIEN T, MURRAY JM, DAVIES KE, PEARSON P. WILLIAMSON R: The use of linked DNA polymorphisms for genotype prediction in families with Duchenne muscular dystrophy. J Med Genet 20:252-254, 1983 PEMBREY ME, DAVIES KE, WINTER RM, ET AL.: Clinical uses of DNA markers linked to the gene for Duchenne muscular dystrophy. Arch Dis Child 5:208-216, 1984