Physical mapping of 14 new DNA markers isolated from the human distal Xp region

GENOMICS

13,167-175

(1992)

Physical Mapping of 14 New DNA Markers Isolated from the Human Distal Xp Region M. C. WAPENAAR,* C. PEm,-f E. BASLER,$ A. BALLABIO,$ A. HENKE,~ G. A. RAPPOLD,~ H. M. B. VAN PAASSEN,* L. A. J. BLONDEN,* AND G. J. B. VAN OMMEN* *Department of Human Genetics, Sylvius Laboratory, Leiden University, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands; tUnite de Recombinaison et Expression Genetique, institute Pasteur, 25 Rue du Dr Roux, 75724 Paris Cedex, France; Slnstitute for Molecular Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030; and Slnstitut fur Humangenetik und Anthropologie, Ruprecht-Karls-Universitat Heidelberg, Im Neuenheimer Fe/d 328, 6900 Heidelberg 7, Germany Received October 7, 1991 revised December 25, 1991

We have isolated 14 new DNA markers from the human Xpter-Xp21 region distal to the Duchenne muscular dystrophy gene by targeted cloning, employing two somatic cell hybrids containing this region as their sole human material. High-resolution physical localization of these markers within this region was obtained by hybridization to two mapping panels consisting of DNA from patients carrying various translocations and deletions in distal Xp. Five markers were assigned to the pseudoautosomal region where their position on the long-range map of this region was further determined by pulsed-field gel electrophoresis. The other nine markers map to the X-specific region. Informative TuqI restriction fragment length polymorphisms were observed for four loci. One of these represents a region-specific low-copy repeated element. These 14 new markers represent useful tools for the understanding of distal Xp deletion and translocation mechanisms and for the positional cloning of disease genes in the region. 0 1992 Academic Press, Inc.

INTRODUCTION

The human Xpter-Xp21 region, measuring approximately 30 Mb, exhibits many interesting genetic features. Proximally, it is bounded by the deletion-prone Duchenne muscular dystrophy (DMD) gene in Xp21, with its size of about 2.4 Mb unparalleled in the human genome (Den Dunnen et al., 1989; Boyce et al., 1991). Distally, it ends in the 2.6-Mb pseudoautosomal region at the extreme terminus of Xp (Petit et al., 1988), which defines a domain of complete homology between the X and Y chromosomes. The entire region is characterized by a high frequency of genetic rearrangements associated with a wide variety of pathology. Illegitimate recombinations between homologous sequences shared between the paternal X and Y chromosomes proximal to the pseudoautosomal boundary (PAB) can relocate the primary sex-determining factor candidate gene SRY (Sinclair et al., 1990) from the Y chromosome to the X

chromosome, resulting in XX male and XY female offspring (Rouyer et al., 1987; Levilliers et aE., 1989). These distal Xp-Yp exchanges are accompanied by deletions in the rearranged chromosomes (Petit et al., 1990a). Abnormal exchanges between homologous regions in Xp and Yq have been found to be involved in patients with X/Y translocations (Ballabio et al., 1989b; Yen et al., 1991). A high frequency of steroid sulfatase gene (STS) deletions (84%) in Xp22.3 is observed in X-linked ichthyosis (XLI) patients (Ballabio et al., 1989c; Yen et al., 1987), and a possible role of flanking repetitive elements in the etiology of the deletions has been proposed (Yen et al., 1990; Ballabio et al., 1990). Extensive deletions affecting adjacent genes on Xp22.3 lead to complex phenotypes known as contiguous gene syndromes (Schmickel, 1986; Ballabio et al., 1989a; Petit et al., 1990a). Complex deletions in Xp22.3 may exhibit clinical features of XLI, short stature (SS), X-linked recessive chondrodysplasia punctata (CDPX), mental retardation (MRX), Kallmann syndrome (KAL), and ocular albinism of the Nettleship-Falls type (OAl) (Ballabio et al., 1989a; Schnur et al., 1989). Deletions and duplications in the DMD gene in Xp21.2 cause more than 65% of the Duchenne and Becker muscular dystrophy mutations (Den Dunnen et al., 1989, and references therein). In addition, complex DMD deletions may affect the adjacent proximal loci of chronic granulomatous disease (CGD), McLeod syndrome (XK), and retinitis pigmentosa (RP) (Francke et al., 1985) or, distally, glycerol kinase deficiency (GKD), X-linked adrenal hypoplasia (AHC) (reviewed by Darras and Francke, 1988), and a vision defect which is clinically indistinguishable from Aland Island eye disease (AIED) (Pillers et aI., 1990) but seemsto be genetically distinct from the original disorder described on Aland Island (Alitalo et al., 1990). The etiology of deletions affecting the DMD gene remains elusive. As yet, no low-copy repeats have been found dispersed in the deletion hot spots of the DMD gene. The deletion proneness of this region might thus rather depend on the local chromosome structure (Blonden et al., 1991).

167

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168

WAPENAAR

Whichever their nature, the numerous deletions and contiguous gene syndromes in distal Xp have allowed the assembly of many valuable deletion panels assisting in the mapping of genes, disease loci, and DNA markers (Ballabio et al., 1989a, 1990; Petit et al., 1987, 1990a-c; Hardelin et al., manuscript in preparation). In parallel, long-range physical maps were constructed from the pseudoautosomal region (Brown, 1988; Petit et al., 1988; Rappold and Lehrach, 1988), Xp22.3 (Ballabio et al., 1990; Petit et cd., 1990b; Li et at., 1990; Ross et aZ., 1990), and Xp21 (Burmeister et al., 1988; Den Dunnen et al., 1989). This has allowed the high-resolution mapping of new markers located in both ends of the Xpter-Xp21 region. Here we present the physical mapping of 14 new DNA markers from the Xpter-Xp21 region isolated by targeted cloning, using two somatic cell hybrids containing this region as their sole human material. These markers were assigned to the deletion intervals of two mapping panels and further located on a pulsed-field gel electrophoresis (PFGE) map of the pseudoautosomal region. This panel of additional probes should contribute to regional mapping, localization, and isolation of genes and disease loci in distal Xp and may help to shed more light on the features of this region as mentioned above. MATERIALS

AND

Cell lines. Somatic cell hybrids al., ISSOa) were cultured in DMEM with 10% fetal calf serum (Gibcol.

and TG2 (Wapenaar (Gibcol supplemented

AL.

XP

IL21 11.l 11.1 113

DE1

FIG. 1. Map of the human DNA content TG5scS.l and TGZ, which were used to clone probes (Wapenaar et al., 1990al.

METHODS

TG5scS.l medium

ET

in somatic cell hybrids and select the distal Xp

et

Deletion panels. The deletion intervals in the mapping panels, defined by breakpoints in the DNA of patients carrying Xp interstitial and terminal deletions as well as patients with Xp deletions associated with abnormal X-Y exchanges, have been described previously (Ballabio et al., 1989a, 1990; Petit et al., 1987, lSSOa-c; Hardelin et al., manuscript in preparation). Probes. Probes were derived by PstI subcloning of pooled DNA from 700 human cosmids which were cloned from hybrid TG5scS.l. Details of cosmid library construction, subcloning and selection of single-copy PstI subclones, and their mapping with respect to the Xp21 breakpoint in hybrid TG2 have been described (Wapenaar et& 1988). PFGE mapping. DNA for PFGE analysis of the pseudoautosomal region was prepared from human peripheral lymphocytes (46, XX) in agarose blocks. Methods of PFGE, digestion, and DNA transfer were as described (Herrmann et al., 1986, 1987). Southern blot analysis. All DNA manipulations involving DNA isolation from cell lines and peripheral lymphocytes, restriction enzyme digestions, blotting, probe labeling, and hybridization were performed according to methods pubiished previously (Petit et aL., 1987; Herrmann et al., 1987; Wapenaar et al., 1988; Ballabio et al., 1989a).

RESULTS

Mapping of Probes We have previously isolated two somatic cell hybrids containing Xpter-Xp21 fragments as their sole human material against a hamster background which are an ideal source for cloning from this region (Wapenaar et al., 1990a). The Xp21 breakpoint of TG5sc9.1 is located proximal to the DMD gene between markers PERT469

(DXS196) and cX5.7 (DXS148), while TG2 lacks the DMD gene and breaks proximal to the nearest DMD flanking marker C7 (DXS28) (Fig. 1). A pool of 700 human cosmids was prepared from TG5sc9.1, and singlecopy PstI subclones were mapped with respect to the breakpoint in TG2. Of 24 single-copy probes tested, 14 could be mapped to the TG2 cell line, one was assigned proximal in Xp21,6 were located in the pericentromeric region, and the remaining 4 clones failed to hybridize or were of hamster origin. The marker that mapped proximal to the TG2 breakpoint, P20 (DXS269), was localized in the middle of the DMD gene where it detects the major, centrally located, deletion hot spot (Wapenaar et al., 1988; Blonden et al., 1989). The 14 single-copy clones that mapped to the TG2 interval (Table l), i.e., distal to the DMD gene, were further localized with respect to the breakpoint of patients with abnormal X-Y exchanges, terminal deletions, and interstitial deletions gathered in two deletion panels described previously: panel I (Ballabio et al., 1989a, 1990) and panel II (Petit et al., 1987, 1990a-c; Hardelin et al., manuscript in preparation). The results of the localization of these probes are summarized in Fig. 2. Five markers were assigned to the pseudoautosomal region. Four of these, P99 (DXYS87), P131 (DXYS86), P9 (DXYS75), and P117a (DXYFSGSl]), map distal to a breakpoint in panel II, which separates loci DXYS59 and DXYS15, whereas P12 (DXYS85) maps proximal to this breakpoint. P117 represents a region-specific low-copy repeat (see below), and a second region with homology, P117b (DXF36S2),

MAPPING

TABLE Characteristics Probe P4 P9 P12 P32 P39 P45 P59 P65 P85 P90 P99 P117a P117b P122 P131 D Determined

OF

14 NEW

DNA

MARKERS

1

of 14 New Loci from the Human Xpter-Xp2 1 Region Locus

Insert

DXS404 DXYS75 DXYS85 DXS408 DXS409 DXS410 DXS411 DXS412 DXS413 DXS414 DXY S87 DXYF36Sl DXF36S2 DXS418 DXYS86 by DNA

in bp

1400 48” 232” 1700 1900 740 119” 4500 940 340 1000 156” 156” 1670” 1000

RFLP

VNTR

TaqI

TuqI VNTR

sequencing.

has been found in interval d of panel I, to which nonspecific mental retardation (MRX) was assigned (Ballabio et al., 1989a). Of the nonpseudoautosomal markers, P39 (DXS409) was localized together with marker DXS3l to two nonidentical but similar deletion intervals in both panels. Both P85 (DXS413) and P59 (DXS411) map to the region of the STS gene involved in X-linked ichthyosis. P85 lies in interval e of panel I. The border between intervals e and f is defined by a partial deletion in the STS gene of a patient with isolated XL1 (patient A.B. in Ballabio et al., 1989a). Most likely, this result maps P85 distal to STS in a region of approximately 400 kb between DXF30Sl (S232a) and STS/DXYS74 (Ballabio et al., 1990). The position of P59 was deduced from both mapping panels as proximal to DXFSSOSl (S232a) and DXFS30S2 (S232b) but distal to DXF30S3 (S232c) in a region of ca. 800 kb containing STS, DXYS74, and DXS237. Probe P4 (DXS404) enables us to make a subdivision in interval k of panel I, since it is absent in patient 15 (J.D.) but present in patient 16 (D.N.) (for nomenclature of these patients, see Ballabio et al., 1989a). This maps P4 in interval k2, proximal to DXS143 in kl. This localization of P4 proximal to DXS143 was confirmed by its mapping to the larger amelogenin gene (AMG)/DXSS interval in panel II. Panel II allows the further division of the remaining five markers located in interval 1 of panel I to two groups. P45 (DXS410) and P90 (DXS414) map distally in the AMG/DXSS interval and P32 (DXS408), P122 (DXS418), and P65 (DXS412) proximally in the ZFX interval, which defines the end of the resolution of panel II. Preliminary mapping of these proximal markers in Xp22 has shown that P32 (DXS408) maps between DXS28 and DXS41 (H. Y. Zoghbi, personal communication). The five markers localized in the pseudoautosomal region were mapped more precisely by PFGE analysis. Three of these have been published previously (Henke et

IN

HUMAN

DISTAL

169

Xp

al., 1991). The fragments obtained with the rare cutter enzymes NotI, BssHII, SC&, iVru1, CM, and EagI are summarized in Table 2. Examples of hybridizations are shown in Fig. 3, together with the positions of the markers on the long-range map of the region. Probe 29A24 (DXYSSO) has been placed 20-30 kb from the telomere (Cooke and Smith, 1986). The smallest fragment shared by 29A24 and P99 is a 420-kb ClaI fragment in addition to common 470-kb NotI, SalI, NruI, and CZaI fragments. The largest fragment unique to 29A24 is a 380-kb ClaI fragment. These results together place P99 380-420 kb from the telomere. Additional data (not shown) place pDP411 (DXYS28) (Page et al., 1987) on our map between 470 and 500 kb. P9, P131, and P117 detect identical bands for all enzymes tested and are all contained within a 170-kb EagI fragment. The additional bands seen for P117 in Fig. 3 are caused by the low repetitive nature of this probe which detects homologous loci elsewhere in distal Xp (see below). Physical linkage between the P9/P131/P117 cluster and probe 113D (DXYS15) (Simmler et al., 1985) was demonstrated by a common 800-kb NotI, an 800-kb BssHII, 260- and 680kb SaZI, 590-, 620-, and 800-kb NruI, 650- and 800-kb Mb

Mb 0

-I

1

-

SS 2 3 CDPX 4 5 6 7 6 9 DXSlQ

l’Q-30 D-

-+DX!MO4 DXS410 DXS414 I-DXS406 I DXS416 DXS412 I-

y

10

DXS9

‘\

,’ ZFX

,/” /20-30

FIG. 2. Summary of the assignment of 14 new DNA markers with respect to the deletion intervals of two Xpter-Xp22 mapping panels. The two mapping panels, consisting of various deletion and translocation patients, together with the long-range map positions of reference markers and gene loci, have been described for panel I in Ballabio et al. (1989a, 1990) and for panel II in Petit et al. (1987,1988,1990a-c). The indices in the deletion intervals refer to the nomenclature in previous publications (Ballabio et al., 1989a, 1990; Petit et aZ., 1990b). The additional intervals in panel II will be described by Hardelin et al. (manuscript in preparation). These represent terminal deletions in males, some of which are associated with Xp/Yq translocations, except for the most proximal breakpoint which is present in a female carrying an isodicentric X. The deletion intervals have not been drawn exactly to scale.

170

WAPENAAR

TABLE Summary Probe 29A24

of PFGE

Mapping

(locus)

Not1

(DXYSBO)

(100)

(80)

150 (230)

140

Results

BssHII

(220)

(280)

ET

AL.

2 in the Pseudoautosomal Sal1

NruI

(50) 90 (170) 470

(180) 250 470

300 470

220 470

(470) P99 (DXYS87)

(120)

250

190

(320) (370) (470)

113D

800

800

800

(DXYS15)

-

ClaI

(300) 340 (380) (420) 470 50 130

(420) 470

(250)

P9 (DXYS75) P117 (DXYF36Sl) P131 (DXYS86)

Region”

260 (680)

590 620

750

170

(260) (350) (610) 700 800

(800)

(800)

260

590

(680)

620 (800)

(340) 430 (470)

1100

180 320 400,430 (740) (790)

a Fragment sizes are in kb. Weak bands (which are considered to represent partials) are in parentheses; Fragments identified by at least two different probes are in boldface type. To confirm identity of bands were run using appropriate resolutions for the different size ranges.

ClaI, and 350-, 610-, 700-, and 800-kb EagI fragments. Fragments specific to 113D were 340-, 430-, and 47O-kb ClaI bands and 90-, 180-, and 440-kb EagI bands and fragments unique to the P9 cluster were a 190-kb ClaI and a 170-kb EagI band. This locates P9/P131/P117 at 500-670 kb from the telomere, distal to 113D at position 670-720 kb (Petit et al., 1988; Brown, 1988; Henke et al., 1991). The probes P12 and 602 (DXYS17) (Rouyer et al., 1986) map within 180 kb according to a shared CZaI fragment located 1700-2000 kb from the telomere. Restriction Fragment Length Polymorphisms Although we did not perform a systematic screening for polymorphisms, we did observe RFLPs for some of the markers in the course of mapping. P99 (DXYS87), with the most distal pseudoautosomal location, detects TaqI alleles of 6.5,5.0, 3.8, and 1.2 kb (Fig. 4a). The 3.8and 1.2-kb bands cosegregate and are separated by a TuqI site mutated in the 5.0-kb fragment. The 6.5-kb band suggests that one of the TuqI sites of the 5.0-kb fragment is also polymorphic and that another TaqI site is 1.5 kb away. This would predict that we might expect to observe in a larger sample size either a 2.7-kb (1.2 + 1.5 kb) or a 5.3-kb (3.8 + 1.5 kb) band. In accord with its

(100) 130 190

190

(800)

900

30 70 120

(650)

(650)

P12 (DXYS85) 602 (DXYS17)

BagI

90

(180) (350) 440 610 700 800

320 (350) (750)

strong bands are without parentheses. recognized by two probes, several gels

pseudoautosomal location, heterozygote males can be observed (Fig. 4a). Allele frequencies are 0.5 for the 5.0kb band, 0.42 for the 3.8/1.2-kb pair, and 0.08 for the 6.5-kb fragment in 24 chromosomes studied. P9 (DXYS75) detects a VNTR with a 0.95 heterozygote frequency (at least 18 TaqI alleles in a 1.5-5.5 kb range) which has been described previously (Wapenaar et al., 1990b). This probe has been extremely useful in our hands for resolving identity and paternity issues in blots of our diagnostic program. P39 (DXS409), which maps proximal to the PAB, detects a frequent TaqI RFLP with a major allele frequency of 0.55 (5.8 kb) and a minor allele frequency of 0.45 (7.6 kb) in 20 chromosomes tested. Distribution of the alleles is compatible with an X-specific probe showing hemizygous males (Fig. 4b). The TaqI hybridization pattern of P117 is quite complex (Fig. 4c) and indicates a low-copy repeat consistent with its localization to at least two deletion intervals of the mapping panel (see above). Most prominent are the variable-length bands between 3.9 and 2.8 kb, which map to the pseudoautosomal region (result not shown). The number of polymorphic bands (two to four) present both in males and in females, and their relative intensi-

MAPPING

OF

14 NEW

DNA

MARKERS

LM-

470420-

50kb P99 oDP411

P131 -r

P117 -T

113D -r

P12/602

P ,“B,

x CM

I

I 4aokb

FIG. 3. Localization of five new DNA probes (P9, P12, P99, Pll7a, and P131) on the long-range map of the pseudoautosomal region. High-molecular-weight DNA isolated from peripheral blood of a female individual (46,Xx A.H.) was digested with SalI, &I, NruI, EagI, and NotI. Electrophoresis was carried out in an LKB Pulsaphor Unit for 42 h and a 45-s pulse time (pDP411 and P99) or for 46 h and a 65-s pulse time (P131, P117, and 113D). Gels (0.9% agarose) were run in 0.25X TBE at an electric field gradient of 7.5 V/cm at 14°C. LM indicates the limiting mobility zone. The lower part of the figure gives a schematic representation of the positions of the five new probes and the previously mapped markers, 29A24 (DXYSZO), pDP411 (DXYS28), 113D (DXYS15), and 602 (DXYSl7), on the physical map of the pseudoautosomal region. The solid black bars indicate the accuracy range of mapping. PABX is the pseudoautosomal boundary on the X chromosome.

ties compared with the constant bands suggest that I?117 detects two adjacent VNTR loci (DXYF36Sl) in the pseudoautosomal region, possibly located within a single 170-kb EagI fragment. In addition, a Y-specific 4.5kb and an X-specific 4.3~kb band can be observed and should, by definition, be localized proximal to the pseudoautosomal boundary. Constant bands of 6.4,2.35, 2.1, 1.7, and 1.45 kb can be seen, whereas longer exposure reveals six fainter additional bands. Comparing the P117 banding patterns of different individuals with that of hybrid TG2 showed that all fragments, except the 4.5kb Y-specific band, are contained within the XpterXp21.3 region, underscoring the localized region-specific nature of this repeat family. No (frequent) RFLPs were observed for the proximal markers P4, P45, and P90, tested with the enzymes EcoRI, TaqI, BclI, HindIII, PstI, RsaI, and PuuI (A.A.B. Bergen, personal communication). DISCUSSION

In this report we present data on the high-resolution mapping of 14 new DNA markers isolated from the

IN

HUMAN

DISTAL

Xp

171

Xpter-Xp21 region. These markers were isolated by targeted cloning and rapidly assigned to the region distal to the DMD gene by employing two somatic cell hybrids whose breakpoints flanked this gene (Wapenaar et al., 1990a). We have used this approach previously to select the probe P20 (DXS269) from the DMD gene which detected the major deletion hot spot (Wapenaar et al., 1988). We now report the localization of 14 probes distal to the DMD region, using two complementary, partially overlapping Xpter-Xp22 deletion panels. These panels were assembled from patients with interstitial and terminal Xp deletions (including abnormal X-Y exchanges), some of them suffering from contiguous gene syndromes. Deletion intervals, as defined by the various breakpoints, were determined previously with partly overlapping sets of markers (Ballabio et al., 1989a; 1990; Petit et al., 1987,1990a-c; Hardelin et al., manuscript in preparation). The assignment of the new markers to these intervals has improved the alignment of the two panels and increased their information content by further subdivision of original intervals. Five markers were mapped to the pseudoautosomal region (PAR). These were subsequently placed on a long-range map of this region by PFGE (Petit et al., 1988; Brown, 1988; Rappold and Lehrach, 1988; Henke et al., 1991). The PAR is characterized by a high recombination frequency, which is suggested to be 1 CM in 55 kb in male meiosis, due to a single obligatory X-Y crossover (Rouyer et al., 1986; Petit et al., 1988). Close physical linkage of our probes to markers previously used in genetic mapping, in principle, would predict the following genetic distances to the telomeric reference marker DXYSBO: P99 at 11 CM, P9/P117/P131 at 16 CM, and P12 at 40 CM (Rouyer et al., 1986; Page et al., 1987). These derived positions on the genetic map should be useful for the polymorphic markers when applied in linkage studies. Genes that have been assigned to the PAR include MIC2 (Goodfellow et al., 1986), XGR (Goodfellow et al., 1987), GM-CSF receptor (Gough et al., 1990), and short stature (SS) (Ballabio et al., 1989a). Short stature, the only consistent feature present in all Turner females, may be caused by the monosomy of the middle portion of the pseudoautosomal region, localization of which was recently further confined with the probes described in this report (Henke et al., 1991). It is striking that the PAR probes presented here are clustered together with previously existing markers. The sparsity of single-copy probes in the ca. lOOO-kb region (40% of the PAR) which separates DXYSlS from DXYS17 and comprises two HTF islands (Petit et al., 1988) either might result from a small sample size or might reflect a biased cloning efficiency for this region. Abnormal Xp-Yp exchanges leading to XX males and XY females are observed at high frequencies within a distance of 4 Mb from the telomere (Levilliers et al., 1989; Petit et al., 1990a,b) and are thought to result from sequence homologies which extend beyond the pseudoautosomal boundary (Petit et al., 1990a). It has

172

WAPENAAR

ET

AL.

c mfffffmmmmfffmmml a

mf

f fmmmffff

f

P99

b

em*

_i

E

*-

- 1.45

P39 P117 FIG. 4. Examples of TaqI RFLPs detected by probes PSS (a), P39 (b), and P117 (c) on DNA samples isolated from independent males (m) and females (f). Detailsare described in the text. (1) Size ladder (mixture of lambda DNA digested with HindUI andPstI). SeaKern) were run for 18 h at 20 V in 1~ TAE.

been hypothesized

that the ancestral PAB was located in Xp22.3 and Yp, on the basis of the following observations: (1) the escape from X inactivation of pseudoautosomal and Xp22.3 genes (Race and et al., Sanger, 1975; Shapiro et al., 1979; Goodfellow 1984); (2) the presence of nonmethylated CpG islands in Xp22.3 in inactivated X chromosomes (Petit et al., 1988, 1990b); and (3) the pseudoautosomal location of the murine STS gene (Keitges et al., 1985). The observed sex chromosome-specific homologous sequences in Xp22.3 and Yp are thought to be remnants of this evolutionary past. The latter group include subtelomeric interspersed repeat (STIR) elements which have a high prevalence in and close to the pseudoautosomal region (Simmler et al., 1985; Petit et al., 1990a). The X-linked chondrodysplasia punctata (CDPX) gene has been mapped, together with marker DXS31, within a distance of 3 Mb proximal to the pseudoautosoma1 boundary (Petit et al., 1990b). Two deletions have been described which place DXS3l proximal to CDPX (Ballabio et al., 1989a; Petit et al., 1990b). P39 maps in the same deletion interval in which DXS31 has been localized, i.e., proximal to CDPX. The informative P39 TaqI RFLP (PIC 0.3725) should thus be a close proximal flanking marker for CDPX linkage analysis and will provide further genetic mapping information on its position with respect to DXS31. DXS31 belongs to the group of sequences in Xp22.3 which has a homologous counterpart on Yq as a result of an alleged pericentromeric inversion in the Y chromosome earlier in evolution (Yen et al., 1988). These Xp-Yq homologies are thought to be involved in the etiology of X/Y translocations (Ballabio et al., 1989b; Yen et al, 1991). The strictly X-specific nature of P39, which is physically closely linked to DXS31, indicates how closely X-specific and X-Y homologous sequences are interdigitated in Xp22.3.

more proximally

Caucasian Gels (0.8%

Close physical linkage to the deletion-prone STS gene was obtained for P59 and P85, but additional PFGE or deletion data are necessary to pinpoint their exact position in the map. The STS locus is flanked by members of the low-copy repeat families DXF30 (S232) and DXF22 (G1.3) (Ballabio et al, 1990). The occurrence of repeatassociated deletion breakpoints suggests a mechanism of unequal recombination between the individual members of these families in the etiology of deletions in this region (Yen et al., 1990; Ballabio et al., 1990). The characteristics of P117-the presence of two adjacent VNTR loci in the pseudoautosomal region, one locus in the MRX gene interval d, the presence of X-Y homologous as well as several Xpter-Xp22-specific copies-indicate that we have cloned a new family (DXYF36) of region-specific low-repetitive DNA which, in addition to the DXF30 and DXF22 families, may play a role in the observed instability in the distal Xp region. Ocular albinism of the Nettleship-Falls type (OAl) was found to cosegregate with a Xp22.3 microdeletion removing the STS gene, which suggested a contiguity of these two loci (Schnur et al., 1989). Recently, however, Schnurr et al. (1991) have revised this assignment, since genetic linkage data map OAl proximally outside this deletion, between DXSl43 and DXS85. A location of OAl proximal to DXS143 would be in agreement with the fact that XLI-KAL deletions do not include OAl, a result that places OAl proximal to the DXS143 interval k (Ballabio et al., 1989a). Hence, the subdivision of interval k by P4 combined with its location proximal to DXS143 would predict a close physical linkage of P4 to OAl. However, genetic mapping data of Bergen et al. (1991) locate OAl with a ca. 100 times greater likelihood distal to DXS143 than proximal to it. This paradox may imply either that DXS143 maps within the OAl locus or that the picture is confused by incomplete penetrance,

MAPPING

OF

14 NEW

DNA

MARKERS

additional Xp rearrangements, or an erroneous genetic order caused by one or undetected double recombination. Further genetic and physical analysis in this region, including the potentially interesting markers P45 and P90, is therefore warranted. Preliminary testing of five independent OAl patients with eight of the potentially interesting markers, P85, P59, P4, P45, P90, P32, P122, and P65, did not show a deletion (result not shown). The markers proximal to DXS143, subdivided in three deletion intervals, P4-(P45, P90)-(P32, P65, Pl22), may contribute to a more detailed mapping of the Xp22.2-Xp22.1 region. This region contains the genes involved in amelogenesis imperfecta (Lagerstrbm et al., 1990), X-linked juvenile retinoschisis (Sieving et al., 1990), hypophosphatemic rickets (Thakker et al., 1990; Econs et al., 1990), Coffin-Lowry syndrome (Hanauer et al., 1988), Nance-Horan syndrome (Stambolian et cd., 1990), spondyloepiphyseal dysplasia tarda (SzpiroTapia et al., 1988), and Aicardi syndrome (Ropers et al., 1982). The availability of cosmids from several of the loci should facilitate deletion mapping by whole cosmid hybridization (Blonden et al., 1989,199l) and the isolation of microsatellite markers (Weber and May, 1989) to extend their information value for genetic mapping. In forthcoming studies, the sequence data of these markers will be used to convert them in STSs (Olson et al., 1989) and to screen YAC libraries to extend the locus size (Green and Olson, 1990). In addition, these markers may contribute to a more detailed physical map of the proximal Xp22 region, both by PFGE and by fluorescence in situ hybridization. Recently we have extended our efforts at targeted cloning of the Xpter-Xp21 region by isolating a YAC panel from hybrid TG5sc9.1 (Driesen et al., 1992; Wapenaar et al., manuscript in preparation). ACKNOWLEDGMENTS This work was supported by grants from the Dutch Prevention Fund, the Medical Science Division of The Netherlands Organization for Scientific Research (NWO-GBMW), and the Muscular Dystrophy Association (MDA) of America to G.J.B.v.0. DFG Grant Ra 380/31 and Grant TG8 of the Ministerium fur Wissenschaft und Kunst Baden-Wiirttemberg to G.A.R. and from the National Institute of Child Health and Human Development to A.B. C.P. was supported by the Ministere de la Recherche et de la Technologie (Grant 9O.C.0518) and by the Association Francaise contre les Myopathies (Grant C.S.22/ 01/90 code 3 CA S/03/90).

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