GENOMICS

11,352-363

(1991)

Physical Mapping

of 60 DNA Markers in the p21 .I + q21.3 Region of the Human X Chromosome

RONALD G. LAFRENI~RE,* CAROLYN J. BROWN,* VICKI E. POWERS,* LAURA CARREL,* KAY E. DAVIES,t DAVID F. BARKER,* AND HUNTINGTON F. WILLARD*,’ *Department of Genetics, Stanford University School of Medicine, Stanford, California 94305; tlnstitute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom; and *Department of Medical Informatics, University of Utah, Salt Lake City, Utah 84108 Received

April

the human X chromosome. Our goals were to establish the physical order of loci in this region, to find new and useful RFLP markers to aid genetic mapping efforts, and to use these data to help examine recombination at and near the human X centromere (Mahtani and Willard, 1933; Mahtani et al., 1991). This region also contains the X inactivation center (Brown et al., 1991b) as well as a number of genes that escape X inactivation (Brown and Willard, 1990; Fisher et al., 1990), making it an important region for studies of this unique regulatory mechanism. In this study, we have physically mapped 60 markers to 15 different intervals defined by X-chromosome rearrangements and deletions isolated in somatic cell hybrids and human lymphoblast lines. The physical ordering of these X-linked loci should help focus other genetic and long-range physical mapping efforts in the proximal regions of both the long and the short arms of the human X chromosome.

Using a panel of human/rodent somatic cell hybrids and human lymphoblast lines segregating 18 different human X-chromosome rearrangements and deletions, we have assigned 60 DNA markers to the physical map of the X chromosome from Xp21.1 to Xq21.3. Data from Southern blot hybridization and polymerase chain reaction (PCR) amplification assign these markers to 15 primary map intervals. This provides a basis for further long-range cloning and mapping of the pericentromeric region of the X chromosome. 0 19S1AcedemicPress,Inc.

INTRODUCTION

The human X chromosome has historically had the densest gene map, with well over 200 genes assigned to it on the basis of their mode of X-linked inheritance (McKusick, 1990). The human X chromosome is therefore a primary focus of assorted genetic and physical mapping efforts. Although certain regions of the X have been extensively mapped (Davies et al., 1990), the pericentromeric region has not yet been as well characterized. Since many disorders, such as Wiskott-Aldrich syndrome, retinitis pigmentosa, Xlinked severe combined immunodeficiency, Menkes syndrome, incontinentia pigmenti, Aarskog-Scott syndrome, Norrie disease, anhidrotic ectodermal dysplasia, congenital stationary night blindness, and Xlinked Charcot-Marie-Tooth neuropathy, map to this region (Davies et al., 1990), it is important to establish the relative order of the available cloned markers mapping to this area. As part of long-term studies to investigate several aspects of the X chromosome and its biology, we have focused our primary efforts on creating a physical and genetic map of loci in the pericentromeric region of

1 To whom

correspondence

should

MATERIALS

AND

METHODS

Hybrid Cell Lines Methods used to construct the hybrids have been described elsewhere (Brown and Willard, 1990; Willard et aZ., 1983). A description of the somatic cell hybrid and human lymphoblast lines used in this study is given in Table 1. Rearranged or deleted X chromosomes in female patients were isolated in somatic cell hybrids to permit direct DNA analysis without the presence of an intact X chromosome. Male cell lines (XL62-02 and TEL26), each containing an interstitially deleted X chromosome, were analyzed directly. All rearranged or deleted X chromosomes were naturally occurring, except those in hybrids tllPP-5A and tllPP-GA; these hybrids were derived from a hybrid containing an intact normal X chromosome by isolating clones that continued to ex-

be addressed.

0888-7543/91$3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

12, 1991

352

PHYSICAL

MAP

OF

Xp21.1q21.3

TABLE List of Hybrid Hybrid cell line A2-4 t86-la-2 t76-2ma-lb SIN176 DUA-1CsAzB DUA-1A L62-3A A62-lA-4b A48-lGaz44 A48-1Fa A63-1A tllPP-GA A68-2A tllPP-5A t4-la-azl W4-1A tAG-1Bazlb t81-azlb TEL26 XL62-02 Ben 3B A50-lAc1.3A

t(X; 21) t.(X, 11) t(X, 15) del(X) t(X; 15) ttx; 15) t(X, 17) t(X, 17) t(X; 11) t(X; 11) t(X; 20) del(X) t(X; 22) del(X) ttx; 14) ttx; 14) idic(Xp) del(X) del(X) del(X) tcx; 2) ttx; 7)

t60-12 AHAllaBl

Normal Normal

Entire Entire

Lines Used in This Study Human parental cell line (Ref.)

X component p21.1 --t qter p21.05 + qter p11.3 + qter pter -+ p22.11::p11.23 pter + ~11.22 p11.22 -4 qter pter + p11.21 p11.21 + qter pter + qll.1 qll.1 + qter q11.2 + qter pter + q13.1b q13 --* qter pter + q13.1* pter + q13 q13 + qter pter + q13::q13 + pter + q13.3 pter + q21.1::q22.1 pter + q21.1::q21.3 q21.3 --* qter’ q21.3 + qter

X X

1

Cell Lines and Human Lymphoblast

Chromosomal aberration

353

REGION

+

pter + +

X only X only

qter qter

qter

FRA (66) GM1695 (28) GM1813 “del(Xp)” (30) DW (63) DUV (63) GM7151 (22) GM7151 (22) GM2859 GM2859 GM7792 (49) Normal female GM4628 (16) Normal female GM0074 (1) GM0074 (1) AG (55) “XXq-” (64) TEL26 (=RvD)(lS) XL62-02 (51) “t(X; 2)” (27) GM1696 (58)

M M M H M M M M M M M M M M M H M M H M

Normal GM0144

M M

female (9)

n Refers to a hybrid cell line with a mouse (M) or hamster (H) background. b Localization of the breakpoint was inferred from Southern or PCR analysis. ’ Although the breakpoint for Ben 3B was published as Xq13 (42), we assign it distal to the XL62-02 the A50-lAc1.3A breakpoint. This is consistent with the reevaluation of the breakpoint by Greenberg

press the AlS9T locus in Xp11.3-~11.23 (Brown and Willard, 1990), but had lost the HPRT gene in Xq26 by deletion (Brown et al., 1989c). Hybrid lines A2-4, DUA-1A and DUA-lCsAzB, and Ben 3B were kindly supplied by R. Worton, T. Shows, and D. Ledbetter, respectively. DNA Probes Details of most probes can be found in the DNA report from the Human Gene Mapping 10 and 10.5 workshops (Kidd et al., 1989; Williamson et aZ., 1990) and were obtained from the sources listed in Table 2. PCR Primers and Conditions For analysis of DNA by the polymerase chain reaction (PCR), 100 ng of DNA was amplified for 30 cycles in a Ericomp twin block thermocycler with Promega Tuq polymerase (conditions as described by Promega). A cycle consisted of a 1-min denaturation at 94°C a 1-min annealing at 54”C, and a 4-min elongation at 72°C. Then, 20 ~1 of each 100~~1 PCR reaction was electrophoresed in 1X TAE (0.4 M Tris/Cl,

Rodent parentala

Hybrid Refs. (77) (15) (1% WI (50) (50)

(7‘3 (72) (43) (74) (43) This paper (11) This paper (13) (73) (11) (11) -

(42) (67),

(72)

(12) (71)

breakpoint in Xq21.1, and proximal to et al. (27), who place it in Xq21.3.

0.013 M sodium acetate, 0.002 M EDTA, pH 8.0) through a 2% agarose gel and visualized under ultraviolet light after staining with dilute ethidium bromide. The primers used for DXS426 were described by Luty et al. (1990). Primers for DXS453 were obtained from the American Type Culture Collection (ATCC, Rockville, MD). A 700-bp AlS9T PCR product (AlSSTl-7DH) was amplified from human DNA using the primer pair 5’-GAGCGGGGACTTTGTCTCCT-3’ and 5’-CTTTGACCTGACTGACGAT-3’, derived from the published human cDNA sequence (Zacksenhaus and Sheinin, 1990). A 350-bp PCR product (CHM4-5RH) specific for the choroideremia (CHM) gene was amplified from reverse-transcribed human RNA using the primer pair 5’-TCAGCCTGTTTGACTGCA-3’ and 5’-ACAGTGCCAGCAGAGGAA-3’, based on the published sequence (Cremers et al., 1990). A human PHKA PCR product (PHKA3-4RH) was amplified from reverse-transcribed human RNA using the primer pair 5’-AATTCACTACTGCCCAGGGCTTCAAC-3’ and 5’-GCTTCAGCTCAGCTGGGTTATAGTAT-3’, selected from the mouse cDNA sequence (Bender and Lalley,

354

LAFRENIERE

ET AL.

TABLE List Locus AlS9T AR ARAFl CCGl CHM MAOA MTHFDLl OATLl OA TL2 OTC PGKl PGKI PI PHKA RPS4X SYP TFE3 TIMP XIST DXSl DXS7 DXS14 DXS18 DXS38 DXS56 DXS62 DXS72 DXS94 DXS106 DXS128 DXS131

Probe AlSSTl-7DH pCMVAR pLAR8-4a B CHM4-5RH A2R/D7 clone 316 huOAT6 huOAT6 pOTC” pSPT-PGK pRI-1.7 PHKAB-4RH pDP1313 GH183-1 pTFE3-1.9 TIMP-3.9X” pGlaX2.2 P8” L1.28” ~58-1’ pX22e pXUT2-1.7 L2.98 MGU22 pX65H7 pXG12 cpX203 cX65.3 cpx21

and Source

2

of Probes

Used

in This

Source

Ref.

Locus

L. Carrel D. Lubahn U. Rapp N. Nishimoto L. Carrel X. 0. Breakefield U. Francke D. Valle D. Valle K. Davies A. Riggs A. M. Michelson C. J. Brown D. Page U. Francke J. Puck H. F. Willard H. F. Willard G. Bruns P. Pearson G. Bruns B. N. White H. F. Willard P. Pearson H. Cooke ATCC ATCC ATCC P. Pearson P. Pearson

This paper (41) (4)

DXS132 DXS133 DXS135 DXS136 DXS146 DXS153 DXS159 DXS162 DXS171 DXS226 DXS227 DXS228 DXS255 DXS306 DXS325 DXS339 DXS345 DXS347 DXS348 DXS356 DXS422 DXS423 DXS426 DXS429 DXS441 DXS447 DXS453 DXS467 DXS469 DXS471 DXZl

(61)

This paper (53) (57) (48) (48) (34) (34) (47) This paper (24)

62) (5) (34) (10) (34) (34) (34)

(56) (69) (34) (34) (34) (34) (31) (31)

o These probes are available from the American Type Culture Collection

1989). Both these products were used in Southern blot analysis to map the CHM and PHKA loci. Southern Blot Analysis Conditions for DNA digestion and electrophoresis were essentially as described (Schmeckpeper et al., 1981). Gels were denatured for 45 min in 0.6 it4 NaCl, 0.5 M NaOH, and neutralized twice for 20 min in 0.6 A4 NaCl, 1 M Tris, pH 7.0. DNA was blotted 12-16 h onto Hybond-N (Amersham) nylon membrane using 20x SSC and uv-crosslinked for 90 s using a Fotodyne transilluminator. Blots were rinsed briefly in 2~ SSC and stored at 4°C in prehybridization solution until needed. Escherichio coli host cells harboring cloned DNA markers were grown in TB (Tartof and Hobbs, 1987) 12-16 h at 37°C and plasmid DNA was prepared according to the alkaline lysis method (Birnboim and Doly, 1979). Plasmid DNA was then digested for 3-16 h with the appropriate restriction enzyme(s), fragments were electrophoresed through an 0.8% low-gel-

Study Probe cpX23 cpx30 cpx93 pXl2g pTAK8a cx37.1 cpX289 cpx73 pX63c p2bC6 plbB4 plaA6 M27B SCRlO pQST24Rl pRX21H3” pRX86Rl pRX87Hl pRX97H5 pRX176Ml cpx210 SB1.8 pXL91BlO x2 pRX214Hl pRX404E2 Mfd66 cpXl2 cpX58 cpX251 pBamX7

Source

Ref.

P. Pearson P. Pearson P. Pearson B. N. White T. A. Kruse P. Pearson P. Pearson P. Pearson B. N. White K. Davies K. Davies K. Davies I. Craig P. Goodfellow D. Barker D. Barker D. Barker D. Barker D. Barker D. Barker P. Pearson K. Davies M. Litt B. Schmeckpeper D. Barker D. Barker ATCC P. Pearson P. Pearson P. Pearson H. F. Willard

(34) (31) (34) (56) (34) (34) (34) (31)

(56) (54) (19) (54) (34) (70)

(21) (3) (3) (3) (3) (3) (31)

(42) i& (3)

(68) (31) (31) (31) (34)

(ATCC).

ling-temperature (Seaplaque) agarose gel, and insert bands were cut out after ethidium bromide staining. Insert DNA was prepared by one of three different methods: (1) the insert-DNA-containing agarose slice was melted and DNA was phenol-chloroform extracted and ethanol precipitated according to standard procedures; (2) gel slices were agarased (Calbiothem) 3-16 h at 37°C and insert DNA was labeled directly, without further purification; or (3) insert DNA was centrifuged out of the agarose slice through glass wool (Heery et al., 1990). For each method, lo30 ng (judged by ethidium bromide staining) of insert DNA was labeled to a high specific activity according to the random-primed method (Feinberg and Vogelstein, 1983) using [32P]dATP. Probe concentrations for hybridizations were routinely l-4 rig/ml hybridization solution (l-5 X 10’ cpm ‘“P/M DNA). Prehybridization and hybridization were performed at 42°C as previously described (Schmeckpeper et al., 1981). Up to eight separate blots were hybridized in the same bag in a total volume of 20 ml of hybridization solution. After hybridization, blots were rinsed in 2~

PHYSICAL

MAP

OF

Xp21.1-q21.3

TABLE

355

REGION

3

Results of Hybridization and PCR Experiments Indicating Presence (+) or Absence (--) of a Marker Signal in a Given Cell Line Cell line

Probe

Locus

-

A2R/D7 pOTC L1.28 pX22e pXUT2-1.7 pleA6

++--+-+-+-++--+-+-+-++--+++--+-+-+-++--+-+ ++--+-+-+--

AlSSTl-7DH pLAR&4a huOAT6 TIMP-3.9X p2bC6 pXL91BlO

+++-+-+-++++-+-+-++ +-+-+-+ +++-+-+-+-+ +-+-+-+ + + - +

DXS146 DXS255

GH183-1 pTFE3-1.9 pTAK8a M27B

+++++-+-++ + + + + ++-+-+-+++++-+

OATL2

huOAT6

+

MTHFDLI DXS14 DXS422 DXS423 DXS429 DXZl

clone 316 p58-1 cpx210 SB1.8 x2 pBamX7

+ + + ++++-+-++-+ +

DXZl

pBamX7

+

AR PGKl Pl DXSl DXS62 DXS106 DXS132 DXS133 DXS135 DXS136 DXS153 DXS159 DXS339 DXS348 DXS453 DXS467 DXS469 DXS471

pCMVAR pRI-1.7 ~6 MGU22 cpX203 cpX23 cpx30 cpx93 PXl25 cx37.1 cpX289 pRX21H3 pRX97H5 Mfd66 cpXl2 1~x58 cpX251

CCC1

B

DXS131 DXS162

cpX21 cpx73

MAOA OTC DXS7 DXSlS DXS38 DXS228

AISST ARAFl OATLl TIMP DXS226 DXS246

SYP TFE3

+ +

+ + +

+ + +

+

+

-

+

-

-

-

+

+

+

+

+

+

+

-

-

+

-

3

+

-

+

-

+

-

-

-

+ +

+

-

-

++-++-+ -

1 -

++

+ -

+

4

-

+

+

-

+ +

+ +

-

-

-

-

+

- + -+-++-- + -

+

+

+

-

-

-

+

-

+

-

+

+

+

-

-

-

+

-

6

- + - + +-+-++

+ +

+ +

-

7

-

+

t-+-+-++ + - + + - + +-+-++-+-++ - + +-+-+-++ + - + - + + - + +-+-+-++ +-+-++ - +

-

-

+ +

-

+ +

-

+

-

+

-

+ + +

-

+

-

+

-

+

5

+ + + + + + + + + + +

+

+ + +

+ + +

+

+ - + +-+-+-++

+

+

-

+

+

-

-

+

-

+

+

-

-

+

+

-

+

-

+ + +

+ + + +

-

+

-

++ +

-

+

++

+

+

+

+

+ ++

+

+

+

+ +

+ +

+ +

+ +

+ +

-

-

+

+

+

-

+

+

+

-

-

+

+

+ +

-

+

-

+

-

-

-+-+-++ +

+

+

-

+++

-

-

8

-

9

-

356

LAFRENIfiRE

TABLE

ET

3-Continued Cell

Locus

Probe

RPS4X PHKA DXS227 DXS306

pDPl313 PHKA3-4RH plbB4 SCRlO

XIST DXS128

pGlaX2.2 cX65.3

PGKl DXS56 DXS171 DXS325 DXS347 DXS356 DXS441

pSPT-PGK L2.98 pX63c pQST24Rl pRX87Hl pRXl76Ml pRX214Hl

DXS346 DXS447

pRX86Rl pRX404E2

DXS72

pX65H7

CHM

CHM4-5RH

DXS94

pXG12

AL.

line

Interval

-

+ f-++-+-+-++++ +-++-+-+-++++-+ +

+ +

+

-

+

+

+

-

-

+

+

+

-

-

+-+--+++++-+-+--+++++-+ + + ++--f--+-+++-+-+--+-+++-+ +

+

-

+

+

-

+

-

+

-

SSC, washed at 65°C to a final stringency of 0.1% SDS, 0.1X SSC, sealed in Saran wrap, and exposed to X-ray film (Kodak XAR-5) with intensifying screen (DuPont Cronex Lightning Plus) at -70°C for 3-6 days.

RESULTS

Division of Xp21.1 --+ q21.3 into 15 Intervals A total of 18 independent X-chromosome breakpoints were analyzed by Southern hybridization and/ or PCR using DNA probes and primer pairs that had previously been localized to the pericentromeric region of the X chromosome (Mandel et al., 1989). Based on these previous assignments, some probes were tested against only a subset of the entire panel (see Table 3). All markers were tested against relevant hybrid lines, as well as control human, hamster, and mouse parental lines. Two independent human X-chromosome-only hybrids (t60-12 and AHAllaB1) were used to verify that the bands being scored were X-linked. The presence or absence of any

+

11

+-+++-

12

+ +

+ +

+-+++-+-++++ - + -+-+++--

+

+ +

-

+

+

-+-------

+ +

+ +

+

+

-

-

+ +

-

+

-

13

14

+ +

10

-

+

-

+

-

+

+

+

15

detectable X-linked marker signal in the mapping panel is shown in Table 3. A small number of these results have been reported previously, but all data are included in Fig. 3 and Table 3 for the sake of completeness. Typical Southern blot and PCR results are shown for several of the loci (Fig. 1). For certain translocation breakpoints, both derivative halves of the X chromosome were isolated in hybrid lines, so that the presence and absence of a signal could be doubly verified. Using our probes, we were able to distinguish all but two pairs of breakpoints: A2-4 and t86-la-2, and t81-azlb and TEL26. Both the A2-4 and t86-la-2 breakpoints map within the DMD gene (Koenig et al., 1987), for which we tested no probes, and thus we did not expect to resolve these. The t81azlb and TEL26 breakpoints, however, both define deletion endpoints. Since we were unable to distinguish these using our limited set of probes, it is possible that both these rearrangements break at a common element that favors deletion. In summary, the 18 independent breakpoints allowed us to subdivide the region from Xp21.1 to Xq21.3 into 15 distinct physical intervals, based on the molecular localization of 60 markers from the region (Table 3 and Fig. 3).

PHYSICAL

MAP

OF

a 1

2

3

4

5

6

7

8

9

1.8

DXS429

6.0

DXS132

DXS227

7.2

DXS94

b 1

2

3

4

5

6

7

8

9

10

11

I2

AlS9T

FIG. 1. Physical mapping of markers against the hybrid panel. (a) Genomic DNA was digested with EcoRI and Southern blot analyzed using probes shown on the right. Approximate band sizes are given (in kb) on the left. The figure is a composite of four separate experiments. Lanes: 1, human; 2, AHAllaBl; 3, A62-lA-4b; 4, L62-3A; 5, A48-1Fa; 6, A481Gaz44; 7, A63-lA, 8, A68-2A, 9, W41A. Probes did not hybridize with mouse or hamster parental cell lines (data not shown). (b) A 700-bp product from the AISST gene was amplified using PCR from cell lines containing the Xp11.3~11.23 region. Lanes: 1, human; 2, AHAllaBl; 3, mouse; 4, hamster; 5, A2-4; 6, t75-2ma-lb; 7, SIN176; 8, DUA-1CsAzB; 9, L623A; 10, A481Fa; 11, tl-la-azl; 12, W4-1A.

Screening of Probes for RFLPs To determine whether some of the probes detected polymorphic sites, we performed Southern blot analysis using DNA from six unrelated females that was cut with the following enzymes: AccI, BamHI, BcZI, B&I, BglII, BstEII, DruI, EcoO109, EcoRI, EcoRV, HindIII, HinfI, MboI, MspI, PstI, PuuII, RsaI, ScaI, S&I, S&I, StuI, Z’aqI, XbaI, and XmnI. Of the probes tested, RFLPs were detected only with cpX23 (DruI) (Lafreniere et al., 1989), cX37.1 (BstEII) (Lafreniere

Xp21.1q21.3

357

REGION

and Willard, 1990a), and cpXl2 (RsaI) (Lafreniere and Willard, 199Ob). A rare (1 of 52 X chromosomes screened) EcoRV variant was found in one extended Venezuelan family with probe B of the CCGl locus. No new RFLP was detected with the following probes: pX22e, pLAR8-4a, p2bC6, GH183-1, X2, SB1.8, cpX210, MGU22, pXlBg, pCMVAR, pPGKlPl-RI-1.7, cpX30, cpX58, cpX251, cpX93, cpX21, cpX73, plbB4, pGlaX2.2, and cX65.3.

Close Physical Linkage of DXS1.59, DXS467, and DXS133 Because the cpX probes isolated by Hofker et al. (1987) were all derived from a pooled set of 100 Xlinked cosmids, it was possible that those mapping to the same interval would have originated from one or more common cosmids. During routine RFLP screens, it was noticed that probes cpX289 (DXS159) and cpX12 (DXS467) hybridized to the same size HindIII, BgZII, EcoRI, and XmnI fragments. Also, cpXl2 and cpX30 (DXS133) shared common BumHI and EcoRV fragments. Moreover, all three probes hybridized to a common 11.2-kb XbuI fragment. Southern blot analysis of double-digested human genomic DNA using these probes allowed us to create a genomic restriction map (see Fig. 2). Our results indicate that the three probes could be mapped in the order cpX289-cpX12-cpX30 within the same XbuI fragment. The polymorphic DXS159 PstI site (indicated in Fig. 2d by an asterisk) was mapped in two homozygous females (data not shown). The polymorphic DXS467 RsuI site presumably lies within the cpXl2 probe, since the probe recognizes either one large fragment (2.6 kb) or two smaller fragments (1.9 and 0.7 kb) that add up to the larger fragment. Since the PstI and RsuI polymorphic sites are about 6 kb apart, their genetic informativeness may be increased by considering DXS159 and DXS467 as a single genetic locus.

Gene Order in the X Pericentromeric

Region

No less than 20 cloned genes and pseudogenes have been assigned to the region Xp21.1 to Xq21.3, and we have been able to order some of these. Since both the A2-4 and the t86-la-2 translocation breakpoints disrupt the DMD gene, the short arm genes that we have mapped must be located proximal to the DA4D locus. The genes for ornithine transcarbamylase (OTC) and monoamine oxidase A (MAOA) mapped distal to the Xp11.3 breakpoint, in t75-2ma-lb, placing them in interval 1. Linkage studies and in situ hybridization data (Ozelius et al., 1988) localize MAOA proximal to OTC. Interval 2 contains the genes for the tissue inhibitor of metalloproteinases (TIMP), the A-raf-1 oncogene (ARAFl), and a cluster

358

LAFRENIERE

ET

AL.

- 2.3 - 2.0

d EXSP _ ..

P*. Ba .

s

P B?? BR

E E

?g

P. E

XS . .

m

cpx30 FIG. 2. Physical map around loci DXS159, DXS467, and DXS133. Human female genomic DNA was double-digested and Southern blot analyzed using cpX289 (a), cpX12 (b), and cpX30 (c). Southern bands common to cpX289 and cpX12 are marked with (*), while those common to cpXl2 and cpX30 are marked with (“). Note that all three probes hybridize to an 11.2-kb X&I fragment. A genomic restriction map (d) shows the relative placement of the three probes. Probe cpX289 hybridized to a 2.0-kb SphI-BumHI fragment, but was not precisely mapped within this fragment. Probes cpXl2 and cpX30 (shown as hatched boxes) could be precisely localized to specific EcoRI-P&I fragments. The polymorphic P&I site recognized by cpX289 is indicated with an asterisk in d. Enzyme abbreviations: E, EcoRI; X, XbaI; S, S&I; P, P&I; Ba, BarnHI; Bg, BglII. The black bar indicates the scale of d. DNA size standards for a, b, and c are shown on the right.

of ornithine aminotransferase (OAT)-related se(Lafreniere et al., 1991). AlSST, quences (OATLl) the gene that complements a murine temperaturesensitive DNA replication defect, also maps to this interval. Kirchgessner et al. (1991) have physically linked the SYNl and ARAFl loci based on the presence of a common polymorphic (AC), repeat located 1 kb downstream of SYNl and in the last intron of ARAFl. Using this polymorphism, they have genetically mapped the SYNl/ARAFl loci proximal to DXS7, but distal to the TIMP and OATLl loci. The gene for properdin P factor (PFC) has recently been linked to the TIMP and DXS426 loci and assigned to interval 2 by Coleman et al. (1991), who have established the order TIMP-PFC-DXS426. Interval 3 contains genes for synaptophysin (SYP) as reported facpreviously (Ozcelik et al., 1990) and transcription tor E3 (TFE3). A second cluster of OAT-related sequences (OATL.2) is the only marker tested that separates the t(X;15) and t(X;17) breakpoints that define

4 (Lafreniere et al., 1991). A pseudogene (MTHFDLl) for the trifunctional enzyme (Bozen et al., 1989) maps to interval 5, closest to the centromere

interval

on the short arm. Proximal long-arm genes that have been mapped in this study include the androgen receptor gene (AR) and a pseudogene of phosphoglycerate kinase 1 (PGKlPl), which map to interval 7. AR and PGKlPl, but none of the other probes in the same interval, are deleted in a patient with X-linked androgen insensitivity (Brown et al., unpublished data). A gene required during the cell cycle Gl phase (CCG1) maps to interval 8, while genes coding for ribosomal protein S4 (RPS4X) and phosphorylase kinase A (PHKA) map to interval 10. From sequence analysis of the RPS4X gene, Fisher et al. (1990) have concluded that RPS4X and DXS306, previously reported by Wiles et al. (1988), are in fact the same locus. A transcript specifically expressed from the inactive, but not the active X chromosome (XIST (Brown et al., 1991a)),

PHYSICAL

MAP

OF

AlS9T

iz7 DXSl8 DXS38 DXS228

iE%: PFC SYNl TMP DXS226 DXS426

srp TFE3 DXS146 DXS255

OATL.2

DXZl i%FL1 DXS323

REGION

8

6 MAOA

Xp21.1q21.3

‘AR PGKlPl

DXS324 DXS422 DXS423 DXS429 DXZl

DXSl DXS62 DXS106 E% DXS135 DXS136 DXS153 DXS159 DXS339 DXS.348 DXS453 DXS467 DX.5469 DXS471

CCGI

DXS131 DXS162

‘PHKA RPS4X DXS227 DXS306

PGKl DXS56 DXSl71 DXS325 DXS347 DXS356 DXS441

‘DXS346 DXS447

’ DXS72

+ Xq21.3 showing loci segregated into 15 different intervals. Cytological FIG. 3. Schematic map of the X-chromosomal region Xp21.1 localization of hybrid breakpoints are shown as solid arrows. Only the proximal deletion breakpoints of SIN176, TEL26, and XL62-02 are indicated. Breakpoints for hybrids tllPP-5A and tllPP-GA have not been determined cytologically and are indicated as dashed arrows. Loci are ordered according to HGM designations within each interval. Loci PFC, SYNl, DXS323, and DXS324 are included in the figure based on previously published data (see Discussion) not shown in Table 3.

maps to interval 11, which has also been defined as the interval containing the X inactivation center (XIC) (Brown et al., 1991b). Finally, the PGKl gene maps to interval 12, and the choroideremia gene (CHM) maps to interval 15. By integrating our data with the previously published mapping data discussed above, we can order cloned pericentromeric X-linked genes and pseudogenes as follows: Xpter-OTC-MAOA-

(ARAFl/SYN,

AlSST,

OATLl,

PFC, TIMP)-

(SYP, TFE3)-OATL2-MTHFDLI-cen(AR, PGKlPl)-CCGl-(PHKA,

RPS4Xb

XIST-PGKl-CHM-Xqter. This gene order is consistent with the relative order of the homologous genes on several subchromosomal regions of the mouse X chromosome (Amar et aZ., 1988; Searle et oz., 1989).

DISCUSSION The data presented here generally agree with previously published mapping information. Assignment of MAOA, DXS7, and DXS228 to interval 1 agrees with the codeletion of these markers reported in several patients with Norrie disease (Bleeker-Wagemakers et al., 1989; Forrest et al., 1987). However, localization of DXS18 to interval 1 disagrees with the assignment of this locus to Xcen-ql2 by Riddell et al. (1986). DXSl8 had been reported to map in the Xp11.4-~22.3 interval (HGM7) in 1984, our data suggest that the earlier assignment was correct. The relative order of some of the loci in interval 5 was reported by Sharp et al. (1990) as being Xpter(DXS429, DXS14)-DXS422-(DXZl,cen), based on duplication of some loci in isodicentric X chromosomes. Dietz-Band et al. (1990) mapped loci DXS323 and DXS324 between the breakpoints of hybrids L62-3A and A48-lFa, therefore localizing them to interval 5 (see Fig. 3). Also, Gorski et al. (1991) have physically ordered some proximal Xp probes as

360

LAFRENIERE

Xpter-DXS323-(DXS14, DXS422, DXS429)(DXZl, ten), using X;autosome translocation breakpoints found in patients with incontinentia pigmenti (IPl). This allows us to define the order Xpter-DXS323-(DXS429,

DXS14)DXS422-(DXZl,

ten)

for some of the probes mapping to interval 5. Mapping of the centromeric alpha satellite locus DXZl to intervals 5 and 6, by both Southern analysis and fluorescence in situ hybridization (M. Mahtani, M. Bedford, and V. E. Powers, unpublished data), indicates that the breakpoint of the X;ll translocation isolated in hybrids A481Fa and A48-lGaz44 bisects the alpha satellite array on the X chromosome. Two breakpoints that apparently map within interval 7 have been described and thus help establish the order of some of the loci in this interval. Based on dosage using a male patient (K-M.) with a duplication from Xq12.2 to Xq21.1, and using a derivative X;9 translocation breakpoint isolated in a somatic cell hybrid, Cremers et al. (1988) were able to order the following interval 7 loci as follows: ten-(DXSl, DXS62, DXS136)-(DXS106, DXS132, DXS133, DXS153, DXS159, DXS467, DXS469, DXS471, PGKIPI)DXS135-Xqter. Codeletion of AR and PGKlPl in a patient with X-linked androgen insensitivity (Brown et al., unpublished data) suggests that these two loci are physically very close. Also, DNA linkage analysis by Imperato-McGinley et al. (1990) suggests that DXSl is proximal to the AR locus, based on one observed recombination event. We have shown that DXS133, DXS159, and DXS467 are within the same 11.2-kb XbaI fragment, and for genetic analysis, may therefore be grouped as one locus. Using the same derivative X;9 translocation breakpoint as Cremers et al. (1988), locus DXS339 has been placed proximal to DXS348 (D. Barker, unpublished). Summarizing all these data, we can define the following order for interval 7 loci:

ET

ever, Cremers et al. (1990) have assigned the gene to Xq21.2 based on the cytological localization of an X;13 translocation breakpoint in a female patient. Further mapping studies will be needed to resolve this discrepancy. In this study, we have made use of X-chromosome deletions and rearrangements, X/autosome translocations, and artificially induced breaks to define 15 chromosomal intervals spanning the region between the DMD gene in Xp21 and band Xq21.3 in the proximal long arm. These intervals are defined by 60 markers and cover an estimated genetic distance of over 30 CM (Mahtani et aZ., 1991). Since the pericentromeric region of the X chromosome contains many disease genes, physical mapping efforts, as represented in this study, should greatly assist interpretation of both linkage studies in families with X-linked inherited diseases and overall long-range mapping efforts in this region. ACKNOWLEDGMENTS We thank M. Mahtani, C. B. Sharp, P. Warburton, G. Greig, and R. Wevrick for helpful discussions and technical assistance. The donation of probes from those listed in Table 2 is gratefully acknowledged. This work was supported by Grants HGO6013 (H.F.W.) and DEFG0288ER60689 (D.F.B.).

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DXS153, DXS348)-Xqter.

The locations of DXS339 and DXS453 in relation to the K.M. duplication breakpoint of Cremers et al. (1988) are unknown. Assignment of the choroideremia gene (CHM) to Xq21.3 is based on its localization distal to the Xq21.3 breakpoint in Ben3B (Greenberg et al., 1991). How-

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PHYSICAL

MAP

OF Xp21.1-q21.3

ing Xp21 and non-random inactivation of the normal X chromosome. Hum. Genet. 67: 115-119. 67. WAYE, J. S., ENGLAND, S. B., AND WILLARD, H. F. (1987). Genomic organization of alpha satellite DNA on human chromosome 7: Evidence for two distinct alphoid domains on a single chromosome. Mol. Cell. Bid. 7: 349-356. 68. WEBER, J. L., KWITEK, A. E., MAY, P. A., POLYMEROPOULOS, M. H., AND mBmR, S. (1990). Dinucleotide repeat polymorphisms at the DXS453, DXS454 and DXS458 loci. Nucleic Acids Res. 18: 4037. 69. WIEACKER, P., DAVIES, K. E., COOKE, H. J., PEARSON, P. L., WILLIAMSON, R., BHATTACHARYA, S., ZIMMER, J., AND ROPERS, H. H. (1984). Toward a complete linkage map of the human X chromosome: Regional assignment of 16 cloned single-copy DNA sequences employing a panel of somatic cell hybrids, Amer. J. Hum. Genet. 38: 265-276. 70. WILES, M. V., ALEXANDER, C. M., AND GOODFELLOW, P. N. (1988). Isolation of an abundantly expressed sequence from the human X chromosome by differential screening. Somat. Cell Mol. Genet. 14: 31-39. 71. WILLARD, H. F. (1983). Replication of human X chromosomes in fibroblasts and somatic cell hybrids: Cytogenetic analyses and a molecular perspective. In “Cytogenetics of the Mammalian X Chromosome,” Part A, “Basic Mechanisms of X chromosome Behavior” (A. A. Sandberg, Ed.), pp. 427-447, A. R. Liss, New York. 72. WILLARD, H. F., DURFY, S. J., MAHTANI, M. M., DORKINS, H., DAVIES, K. E., AND WILLIAMS, B. R. G. (1989). Regional

73.

74.

75.

76.

77.

78.

REGION

363

localization of the TIMP gene on the human X chromosome. Hum. Genet. 81: 234-238. WILLARD, H. F., ANLI HOLMES, M. T. (1984). Sensitive and dependable assay for distinguishing hamster and human Xlinked steroid sulfatase activity in somatic cell hybrids. Hum. Genet. 68: 272-275. WILLARD, H. F., AND RIORDAN, J. R. (1985). Assignment of the gene for myelin proteolipid protein to the X chromosome: Implications for X-linked myelin disorders. Science 230: 940-942. WILLARD, H. F., SMITH, K. D., AND SUTHERLAND, J. (1983). Isolation and characterization of a major tandem repeat family from the human X chromosome. Nucleic Acids Res. 11: 2017-2033. WILLIAMSON, R., BOWCOCK, A., KIDD, K., PEARSON, P., SCHMIDTKE, J., CHAN, H. S., CHIPPERFIELD, M., COOPER, D. N., HEWI~, J., LEWI?-~ER, F., MAIDAK, B., Qurrr, M., RICCIUTI, F., AND TRACK, R, (1990). Report of the DNA committee and catalogues of cloned and mapped genes and DNA polymorphisms: Human Gene Mapping 10.5. Cytogenet. Cell Genet. 65: 457-778. WORTON, R. G., DUFF, C., SYLVESTER, J. E., SCHMICKEL, R. D., AND WILLARD, H. F. (1984). Duchenne muscular dystrophy involving translocation of the dmd gene next to ribosomal RNA genes. Science 224: 1447-1449. ZACKSENHAUS, E., AND SHEININ, R. (1990). Molecular cloning, primary structure and expression of the human X linked AlS9 gene cDNA which complements the ts AlS9 mouse L cell defect in DNA replication. EMBO J. 9: 2923-2929.