Murine X-linked genes corresponding to the human Fragile. X (FMRl) and Hunter syndrome (IDS) loci have been mapped in an interspecific backcross between ...
GENOMICS
12,814-817
(19%)
Genetic Mapping on the Mouse X Chromosome of Human cDNA Clones for the Fragile X and Hunter Syndromes CYNTHIA J. FAUST, ANNEMIEKE J. M. H. VERKERK,” PETER J. WiLsoN,t C. PHILLIP Mows,t JOHN J. HOPWOOD,t BEN A. OOSTRA,$ AND GAIL E. HERMAN’ Institute for Molecular Genetics and Department of Pediatrics, Baylor College of Medicine, Texas 77030; *Department of Clinical Genetics and *Department of Ceil Biology, Erasmus University, Rotterdam, The Netherlands; and tlysosomal Diseases Research Unit, Department of Chemicat Pathofogy, Adelaide Children’s Hospital, North Adelaide, Australia Received
August
15, 1991;
Two human X-linked disorders associated with mental retardation, Fragile X syndrome (FRAXA) and Hunter syndrome or iduronate sulfatase deficiency (IDS), map to distal human Xq27. Linkage studies suggest a gene order of FS-FRAXA-IDS-GABRA3-F8 in CEPH and Fragile X families (Le Guern et al., 1990; Suthers et al., 1991a,b,c). The cDNAs for loci corresponding to these two syndromes have recently been cloned (Wilson et aZ., 1990; Verkerk et al., 1991). This region of the human X chromosome is homologous to a region of the mouse X chromosome that lies between factor 9 (Cf-9) and the mouse X-linked visual pigment locus (Rsvp). While more than 100 loci have been mapped on the mouse X chromosome (summarized in Brown et al., 1991), there are no mapped probes in the approximately ~-CM interval between Mcf-2 and Gabra3, loci that lie 1 CM distal and 2 CM proximal to Cf-9 and RSVP,respectively. Using human cDNA probes for the Fragile X (FMRl) and iduronate sulfatase (IDS) loci, we have mapped the corresponding murine loci to the interval between Mcf-2 and Gabra3. Gene order is conserved between the spefor Moof Med-
814 Inc. reserved.
11, 1991
MATERIALS
INTRODUCTION
osss-7543/92 $3.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
October
ties. Since the mouse does not express a fragile site at this location, isolation of the mouse Fmr-1 locus and comparison of the human and murine gene organization could shed light on the mechanism of fragile site production.
Murine X-linked genes corresponding to the human Fragile X (FMRl) and Hunter syndrome (IDS) loci have been mapped in an interspecific backcross between BGCBA-AwmJ/A-Bpa and Mus spretus using human cDNA clones. Pedigree analysis of recombinants from a total of 248 backcross progeny favors a gene order of (Cf-9, Mcf-2)-(Fmr-I)-Ids-Gabra3-Rsvp. Gene order is conserved between the species, although no fragile site has been detected in the mouse in this region of the 0 1992 Academic PWS, IN. UUWiUe X chromosome.
1 To whom correspondence should be addressed at Institute lecular Genetics and Department of Pediatrics, Baylor College icine, One Baylor Plaza, S911, Houston, TX 77030.
revised
AND
METHODS
Mouse strains and crosses. An interspecific backcross between BGCBA-AweJ/A-Bpa and Mus spretus has been described previously (Herman et al., 1991). It consists of a regional mapping panel of 120 animals with recombination events spanning the mouse X chromosome and 11 animals with single recombination events between Cf-9 and Gabra3 in 248 total backcross progeny. The DNA probes used in DNA probes and Southern hybridization. this study and their sources, as well as the hybridization and washing conditions employed for each probe, are listed in Table 1. Southern blotting was performed as described (Herman et aZ., 1991) using Sure Blot Hybridization membranes (Oncor).
RESULTS
Restriction fragment length variations (RFLVs) between M. domesticus and M. spretus have previously been described for the probes Cf-9 and Gabra3 using the restriction enzymes BamHI and TaqI (Buckle et al., 1989; Herman et al., 1991). A BgZII RFLV for the Mcf-2 probe (Grant et al., 1990) was not detected in our cross; however, screening DNA from (C57BL/6JAwpJ X CBA)F, males and M. spretus males digested with several restriction enzymes revealed an MspI RFLV (Fig. 1A). Similarly, for the probes BC22 and pc2S15 detecting murine loci corresponding to FMRl and IDS, respectively, RFLVs were developed using the restriction enzymes PstI and MspI (Figs. 1B and 1C). A TuqI RFLV was also found for the pc2S15 probe, but was more difficult to score. The murine Fmr-1 and Ids loci were regionally localized using representative animals from the interspecific
MAPPING
Fmr-1
AND Ids ON THE MOUSE X CHROMOSOME TABLE
DNA Probes Probe
Locus
F9 (human cDNA) pMT1 (mouse partial cDNA) BC22 (human partial cDNA) pc2S15 (human partial cDNA) pbGRa3 (bovine cDNA)
Cf- 9
815
1
Used in This Study Enzyme detect
Mcf-2 Fmr- 1 Ids Gabra3
used to RFLV
Wash conditions“
Reference Herman et al. (1991) Grant et al. (1990) Verkerk et al. (1991) Wilson et al. (1990) Buckle et al. (1989)
BamHI MspI PstI MspI TaqI
a All hybridizations were performed at 42°C in 40% formamide, 1 M NaCl, 10% dextran sulfate (Pharmacia), 1% SDS, and 200 fig/ml denatured herring sperm DNA. Wash conditions were as follows: A, one wash with 2X SSC, 0.1% SDS at 65°C; one wash with 1X SSC, 0.1% SDS at 65°C. B, three washes with 3X SSC, 0.05% SDS at 65°C. C, three washes with 3X SSC, 0.1% SDS at 6O“C. All washes were performed for 30 min. 1X SSC is 0.15 M NaCl, 0.015 M Na citrate, pH 7.0.
backcross with recombination events spanning the entire X chromosome (not shown). Finer mapping employed the 11 previously identified recombinants between Cf-9 and Gubru3 (Table 2 and Herman et al., 1991). We had not previously mapped Mcf-2, but it had been positioned by Grant et al. (1990) approximately 1 CM distal to Cf-9. As shown in Table 2, it cosegregates with Cf-9 in our cross. Pedigree analysis of the 11 recombinants minimizing the number of crossover events suggests a gene order of (Cf-9, Mcf-2)-(Fmr-l)-Ids-G&a3 for the loci studied. Estimates of the genetic distances between the loci are presented in Table 3. The distance estimates assume that all crossover events between Cf-9 and Gab& were detected in the 248 animals and that no double crossovers have occurred. Since these two loci are approximately 4-5 CM apart (Herman et al., 1991) and double crossovers are extremely rare due to positive interference, these assumptions are likely valid. Similar approaches to genetic mapping have been used by other investigators (see Mullins et at., 1990; Brockdorff et al., 1991). A
DISCUSSION
While conservation of X-chromosomal linkage exists among mammals, gene order is not always preserved. In particular, there must be at least five chromosomal rearrangements between the X chromosomes of human and mouse to account for the gene order in the two species (Davisson, 1987; Amar et al., 1988). As more loci are mapped in both species, boundaries defining homologous blocks become better defined. It appears that within each such block, few, if any, rearrangements have occurred. A large region of conserved linkage extends from Hprt through Rsup on the mouse X chromosome and corresponds to loci mapping to human Xq26-Xq28. The recent isolation of a human iduronate sulfatase cDNA and a partial cDNA for the Fragile X locus enabled our present murine mapping studies extending the number of conserved genes localized to this region. Pedigree analysis of recombinant interspecific backcross progeny strongly supports a gene order of (Cf-9, Mcf-2)-(Fmr-I)-Ids-Gabra3, whichis thusconservedbeP Y
Mcf-2
Ids
5.6 4.2 -
2.5 2.3 -
FIG. 1. Restriction fragment length Table 1. B, male with C57BL/6JAUeJallele; listed to the left of each panel.
variation identified at the Mcf-2 S, male with M. spretus allele;
(A), Fmr-1
s”
(B), or Ids (C) locus. Restriction enzymes B/S, heterozygous N, female. The sizes of fragments
used are listed in (in kilobases) are
816
FAUST
tween human and mouse. Grant et al. (1990) detected a single recombinant in 203 interspecific backcross progeny, placing Mcf-2 0.5 f 0.5 CM distal to Cf-9. In humans, the corresponding MCF-2 locus is within 65 kb of the factor 9 gene (Anson et al., 1988). Comparisons of the distances between these loci in human and mouse are difficult because of extremely high genetic recombination frequencies in human Xq27-Xq28. The distance between Cf-9 and Rsvp is estimated to be 6-8 CM in the mouse (Disteche et al., 1989; Herman et al., 1991) and over 20 CM in the human (Suthers et al., 1991a). Recent fine genetic mapping studies in human Xq27-Xq28 suggest a gene order of F9-(0.19)-FRAXA-(0.015)-IDS-(0.012)-DX304, where the numbers in parentheses are approximate recombination fractions (Suthers et al., 1991a,b). DXS304 is an anonymous probe located approximately 3 Mb proximal to GABRA3 (Poustka and Lehrach, 1991). The recombination fraction between FRAXA and a locus within 800 kb of GABRA3 (DXS374) is 0.15 (Bell et al., 1989). No genetic mapping directly using GABRA3 as a probe has been performed. The Fragile X syndrome derives its name from an association with a rare fragile site at Xq27.3 in affected individuals (for a review see Nussbaum and Ledbetter, 1989). From the sequence of cDNA and genomic FMRl clones, it appears that differential methylation and increased size of a (CGG), repeat contribute to fragile site expression and, probably, to the clinical phenotype (Kremer et al., 1991; Oberle et al., 1991; Verkerk et al., 1991). Fragile sites can be induced on the mouse X chromosome in bands XA2, XC/D, and XFl using agents such as aphidicolin and 5-azacytidine (Djalali et al., 1987,199O; Elder and Robinson, 1989). Based on in situ hybridization, Cf-9 lies in band A6, and the locus Llcam, which is approximately 4 CM distal to Fmr-1 (Herman et TABLE
2
Pedigree
Analysis of Interspecific Backcross Progeny Demonstrating Recombination Events between Cf-9 and Gabra3
Animal
Sex
136 339 397 216 261 159 309 331 350 375 362
F F M F M F F M F M F
(Cf-9, B B S S B S B S B B S
McfB B S S B S B S B B S
2)
Fmrx x x
S S B S B S B S B B S
1
Ids
GU~FU~
S S B
S S
xB XS xB xS xB xS XS S
x
B B S B S B S S B
Note. All recombinants were tested at flanking anchor loci (Ott, Hprt, Pgk- 1, and Ags) and no double recombinants or inconsistencies were detected (Herman et al., 1991). B, C57BL/6JA”-J allele; S, M. spretus allele inherited from the F, heterozygous female. Recombination between loci is indicated by “x”.
ET
AL.
TABLE Recombination and Gabra3 Locus Cf- 9 Fmr- I Ids
3
Frequencies for Loci between on the Mouse X Chromosome
Fmr-
1
Ids
1.2 + 0.7
4.0 + 1.3 2.8 + 1.1
Cf-9
Gabra3 4.4 f 1.3 3.2 -t 1.1 0.4 -c 0.4
Note. Recombination frequencies are calculated in centimorgans f standard error. Estimates of recombination frequency are based on the assumption that all crossover events have been identified by analysis of anchor loci in the entire backcross (Herman et al., 1991). Because no recombinants between Cf.9 and Mcf-2 were identified, Mcf-2 is not included in the table.
al., 1991), maps to band A7/B (Disteche et al., 1989; Brown et al., 1991). The murine Fmr-1 locus should map to band A6/7, which is not associated with a fragile site. Isolation and sequencing of the murine Fmr-1 gene may help to shed light on the mechanism of fragile site formation in human Xq27.3. ACKNOWLEDGMENTS The authors thank Drs. David Nelson and Maura Pieretti for providing the BC22 subclone of FMRl to us and Sylvia Perez for typing the manuscript. G.E.H. is a Lucille P. Markey Scholar and this work was supported by grants from the Lucille P. Markey Charitable Trust and the Baylor Mental Retardation and Child Health Research Centers (5 P30 HD 24064 and 1 P30 HD 27823). C.J.F. was supported by a predoctoral training grant from the National Eye Institute (lT32-EY07102-3).
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MAPPING
Fmr-1
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ON
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