GENOMICS

7, 12-18

(1990)

Establishment of the Mouse Chromosome 7 Region with Homology to the Myotonic Dystrophy Region of Human Chromosome 19q J. S. CAVANNA,* A. J. GREENFIELD,* K. J. JOHNSON,**’ A. R. MARKs,ta$ B. NADAL-GINARD,t’§ AND 5. D. M. BROWN*** *Department of Biochemistry and Molecular Genetics, St. Mary‘s Hospital Medical School, Norfolk Place, London W2 lPG, United Kingdom; and TLaboratory of Molecular and Cellular Cardiology, §Howard Hughes Medical Institute, Childrens Hospital, and *Cardiovascular Division, Bingham and Women’s Hospital, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02175 Received

October

6. 1989;

revised

INTRODUCTION

Myotonic dystrophy (DM) is the commonest form of adult muscular dystrophy and is inherited in an autosomal dominant fashion. The incidence of this diseasehas been estimated in a number of populations as being between 1 in 7000 and 1 in 15,000 (Todorev et al., 1970; Harper, 1989). In all populations, including the Japanese (Takemoto et aZ., 1990), the disease seg’ Current address: Department of Anatomy, Charing Cross and Westminster Hospital Medical School, St. Dunstan’s Road, London W6 SRF, UK. ’ To whom correspondence should he addressed.

12 Inc. reserved.

26, 1989

regates as a single locus on chromosome 19q13.2 and a number of genetic markers tightly linked to DM have been identified. Both physical and genetiq mapping studies have elucidated the order of these markers and show that all of the most tightly linked markers are clustered on the proximal side of DM (Brunner et al., 1989; Johnson et aZ., 1989; Korneluk et aZ., 1989). These flanking markers define the genetic interval on human chromosome 19 into which DM maps. Despite a large number of physiological studies there is still no obvious candidate gene for DM (Renaud, 1987; Harper, 1989), and two possible candidate loci have recently been excluded genetically (Harley et aZ., 1988; Johnson et al., 1988). This lack of an assay for potential candidate genesis a problem that looms larger as the reverse genetic approach to DM gets ever closer to its target. For this reason the mouse chromosomal region with homology to the DM region of human chromosome 19 has been mapped genetically to determine the map position of the mouse homolog of DM. A second important human genetic disease locus, susceptibility to malignant hyperthermia (MHS), has been shown to map to chromosome 19q13.2/13.1 (McCarthy et al., 1990). A candidate gene in humans for the MHS locus, the ryanodine receptor (RYR), has recently been cloned from a rabbit cDNA library (Takeshima et al., 1989; Marks et al., 1989). The MHS locus has been shown to be closely linked to markers in the region of 19q13.2113.1 and is tightly linked to the marker CYPBA, emax= 0, Z,, = 5.7 (McCarthy et al., 1990). We show in this work that the mouse homolog of RYR (Ryr) is located in a position compatible with further consideration for studies as a candidate gene for MHS. In addition, Ryr forms part of a large syntenic group on proximal mouse chromosome 7 that is highly conserved with the myotonic dystrophy region

A number of genetic markers, including ATPlA3, TGFB, CKMM, and PRKCG, define the genetic region on human chromosome 19 containing the myotonic dystrophy locus. These and a number of other DNA probes have been mapped to mouse chromosome 7 utilizing a mouse Mwr domesticus/Mus spretus interspecific backcross segregating for the genetic markers pink-eye dilution (p) and chinchilla (cc’). The establishment of a highly syntenic group conserved between mouse chromosome 7 and human chromosome 19s indicates the likely position of the homologous gene locus to the human myotonic dystrophy gene on proximal mouse chromosome 7. In addition, we have mapped the muscle ryanodine receptor gene (Ryr) to mouse chromosome 7 and demonstrated its close linkage to the Atpa-2, Tgfb-I, and Ckmm cluster of genes. In humans, the malignant hyperthermia susceptibility locus (MI-IS) also maps close to this gene cluster. The comparative mapping data support Ryr as a candidate gene for MHS. o 1990 Academic press. IW.

088%7543/90 $3.00 Copyright 0 1990 by Academic Press, All rights of reproduction in any form

December

MOUSE

CHROMOSOME

7 AND

HUMAN

of human chromosome 19q. The conservation of the genetic map on proximal mouse 7 and human 19q indicates the liI ely positions of both the mouse DM homolog (Dym) and a mouse locus homologous to MHS. MATERIALS

AND

METHODS

Genetic Crosses Interspecific crosses were established as previously described by Greenfield and Brown (1987). Female Mus domesticus homozygous for the pigmentation mutations pink-eye dilution and chinchilla (pcch/pcch) and male Mus spretus (++/++) were mated to yield Fi progeny. F1 females (pcch/++) were backcrossed to male double recessive M. domesticus (pcch/pcch). Some 86 backcross progeny were recovered in this manner and were visually scored for p and cchgenotypes prior to sacrifice and DNA analysis.

DNA Extraction,

Restriction and Southern Analysis

DNA was prepared from backcross progeny and control M. domesticus and M. spretus mice from either tail (Grosschedl et al., 1984) or liver tissue (Brown and Dover, 1979) and digested with a variety of restriction enzymes, fractionated on 0.8% agarose gels, and transferred to Hybond-N membranes (Amersham International). All probes were hexamer labeled (Feinberg and Vogelstein, 1983) and filters were hybridized at 65°C in 6X SSC, 1% (w/v) NaDodSOl, 100 pg/ml denatured, sonicated salmon sperm DNA, 10% (w/v) dextran sulfate, 10 mM Tris, pH 8, and 1 mM EDTA (1X SSC = 0.15 M NaCl, 0.015 M Naa citrate). Filters were washed at various stringencies between 3X SSC and 0.1X SSC, 0.1% NaDodSO* at 65’C and subsequently autoradiographed with Fuji X-ray film at -70°C with intensification for 1 to 4 days.

D 4

SSSD 5455 5658

S D 42 55

CHROMOSOME

13

HOMOLOGY

Probes Atpa- (human locus, ATPlA3) is a 1.2-kb EcoRI/ StuI fragment containing the 5’portion of the rat cDNA from the a-subunit of Na+,K+-ATPase (Kent et al., 1987). Ckmm (CKMM) is a 3.1-kb human genomic clone containing the 3’ portion of the creatine kinase muscle form gene kindly donated by B. Wieringa, University of Nijmegen, Netherlands. Myod is a 1.8-kb cDNA encoded by the murine Myo Dl gene which is involved in myoblast differentiation (Davis et aZ., 1987) and was kindly donated by R. Lassar, Fred Hutchinson Cancer Research Center, Washington. Pkcc (PRKCG) is a 1.4-kb human cDNA clone encoded by the protein kinase C gamma gene and was kindly provided by Dr. Ullrich, Genentech Inc. p134A is a 1.7-kb Sac11 subclone of a cosmid (D19S51) mapping to human chromosome 19 and shows conservation across a variety of mammalian species (Johnson et al., manuscript submitted). Ryr (RYR) is a 700-bp EcoRI subclone of the cDNA encoded by the rabbit ryanodine receptor gene (Marks et al., 1989). Tgfb-1 (TGFB) is a full-length cDNA encoded by the murine transforming growth factor p1 gene and was provided by R. Derynck (Genentech). RESULTS

Analysis of Genetic Crosses An interspecific M. domesticus/M. spretus backcross (seeMaterials and Methods) segregating for the marker loci p and cCh)yielded a total of 86 progeny mice and has been described previously (Greenfield and Brown, 1987). Progeny mice were visually scored for p and cch genotypes prior to sacrifice and DNA analysis. Digests of liver DNAs from backcross progeny mice were hybridized with a number of mouse chromosome 7 and human chromosome 19 probes (see Materials and Methods for details of DNA probes). Figure 1 demonstrates the segregation of M. domesticus and M. spretus restriction fragment length vari-

SSSD 54555658

5

t5

t6

if8

SDSD 5455 56 58

15kb 9.5kb S w 4.4kb 4.2kb Bgl II

D*

8kb SW

3.8kb D +

Ckmm FIG. with a RFLV is also

llkb

D19SSlh Hind III

M-1 Born HI

JW Hind III

PkIX Bgl II

1. Segregational analysis of a variety of mouse chromosome 7 and human chromosome 19q DNAprobes. Progeny DNAs were digested variety of restriction enzymes and analyzed with DNA probes (see Materials and Methods and Table 1). In each case a M. spretus (S) can be observed and these have been indicated. Where the pattern of hybridization is simple, the size of the M. domesticus (D) RFLV shown.

14

CAVANNA

ET

1

TABLE

Probe Probe Atpa(ATPlA3) Ckmm (CKMM) Ckmm (CKMM) Ryr (RYR) Myod Pkcc (PRKCG) D19S51h (D19S51) T&b-l (TGFB)

Enzyme

TagI BglII MspI HindIII BgZII B&II HindIII BamHI

RFLVs

AL.

in M. domesticus

and M. spretus

Diagnostic spretus RFLV 6 4.4 4.8 8 12 5.3 3.5 1.6

’ Following digestion with the stated restriction enzyme, the probes not present in M. domesticus and this table shows their estimated sizes. M. spretus RFLVs (see Fig. 1). Where the pattern of bands detected RFLV(s) is also given. For other probes, M. domesticus has a complex also). Human locus names are indicated where such names have been

ants (RFLVs) in backcross progeny for a variety of probes, and Table 1 shows the band sizes detected by each probe in M. domesticus and M. spretus DNAs following digestion with the stated enzyme. By scoring for the presence or absence of the M. spretus RFLVs detected by mouse chromosome 7-specific probes in individual backcross progeny, it is possible to generate a recombination map (see Table 2 and Figs. 2 and 3) of the proximal portion of mouse chromosome 7. Pedigree Analysis The genetic order of loci was deduced by a simple multipoint or pedigree analysis taking into account the minimum number of crossovers for a given probe order. The order of loci on the proximal portion of mouse chromosome 7 is clearly demonstrated by the pedigree analysis in Fig. 2. Analysis of mice 4 and 42 indicates that D19S51h maps distal to Pkcc and analysis of mouse 55 indicates that Ckmm maps distal to D19S51h, thus placing D19S51h between the Ckmm and the Pkcc loci. However, no recombinants were observed between Ckmm, Tgfb-1, and Atpa-2, indicating that these loci are genetically clustered (see Table 2) on mouse chromosome 7. Mouse 19 places Ryr distal to the Ckmm/ Tgfb-l/Atpa-2 cluster. Mice 32 and 61 demonstrate that Myod maps distal to Ryr but proximal to p. The locus order is thus determined as ten-Pkcc-DlSS5lhCkmm/Tgfb-I/Atpa-2-Ryr-Myod-p-cch. Only two double crossovers were observed (mouse 8 and mouse 24) among the recombinants in the region cchto Pkcc (see Fig. 2 and Table 3). In total, 46 nonrecombinant chromosomes were observed and 38 chromosomes demonstrated single crossovers. A computer printout of the full haplotypes in all 86 mice scored is available on request.

kb kb kb kb kb kb kb kb

DNAs“ domesticus RFLV

2.7, 2.6 kb Many bands Many bands 11 kb 11 kb Many bands 3.8 kb Many bands

mapped in this cross each Progeny mice were scored by any probe is relatively pattern of bands, many of reported.

Wash

conditions

3x 0.1x 0.1x 1x 3x 3x 0.1x 3x

ssc ssc ssc ssc ssc ssc ssc ssc

recognize a M. spretus-specific RFLV that is for the presence or absence of the diagnostic simple, the size of the major M. domesticus which are shared with M. spretus (see Fig. 1

DISCUSSION

This paper describes the mapping of human chromosome 19q markers to mouse chromosome 7 utilizing an interspecific M. domesticus/M. spretus backcross segregating for the mouse chromosome 7 anchor loci p and cch.Recombinant backcross progeny have allowed us to determine both the genetic order and the genetic distance between probe loci on mouse chromosome 7. Some of the described loci have been previously reported as mapping to proximal mouse chromosome 7 (Davisson et al., 1989). Atpa- was reported to be tightly linked to Cyp2b on proximal mouse 7 using an interspecific cross (Kent et al., 1987) and our mapping is in general agreement with this localization. In addition, it has been reported, also using an interspecific cross (Saunders and Seldin, 1989), that two other genes we have examined (Ckmm and Pkcc) are closely linked to Atpa- on proximal mouse 7. These studies have begun to indicate a close homologous relationship between proximal mouse chromosome 7 and human chromosome 19q. Myod, whose human homolog maps to human chromosome llp, was included in this study in order to provide a further marker distal to Ryr and aid in confirmation of genetic order by pedigree analysis (see Fig. 2). Figure 3 shows a comparison of our genetic map of mouse chromosome 7 from Ryr to Pkcc and the established genetic map of human chromosome 19q. It can be seen that the probe order has been inverted with respect to the centromere but otherwise conserved between the two species’ chromosomes. There are no data available for the order of these specific loci on mouse chromosome 7 from intraspecific crosses. However, where comparisons are available the order of loci derived from interspecific crossesis identical with that derived from intraspecific crosses; differences in gene

MOUSE

CHROMOSOME

7 AND

HUMAN

TABLE Recombinational

Analysis domesticus/hf.

D19S51h Pkcc D19S51H Ckmm

2/55 3.6 2 2.5

Ckmm 3/61 4.9 + 2.7 1145 2.2 + 2.2

CHROMOSOME

2

of Mouse Chromosome 7 Markers spretu8 Interspecific Cross@

Tgfb-1 3/78 3.8 zk 2.1 l/54 1.9 * 1.9 O/61 -

Tgfb-1

Atpa3180 3.8 + 2.1 l/55 1.8 +_ 1.8 O/61 o/79 -

Atpa-

15

HOMOLOGY

Ryr 5/63 1.9 + 3.4 2/50 4 + 2.8 l/53 1.9 f 1.9 3/62 4.8 + 2.1 3/63 4.8 iz 2.2

Ryr

in the M.

MY&

P

24/V 31.2 2 5.3 12/53 22.6 + 5.7 15/60 25 f 5.6 21/76 27.6 f 5.1 21178 26.9 k 5 12/61 19.7 ? 5.1

28/80 35.0 + 5.3 16/55 29.1 f 6.1 18/61 29.5 + 5.8 25/80 31.3 f 5.2 25/82 30.5 + 5.1 16/63 25.4 f 5.5 3178 3.8 f 2.1

MY& P

a Progeny from a M. dome&us/M. spretus interspecific cross genotypes of individual progeny mice for p and cch were elucidated of spretm RFLVs identified by a variety of chromosome 7 probes unner out of total number of _- figure - is the number of recombinants recombination fraction in centimorgans with standard error.

CCh

36/80 + 5.6 23/55 41.8 + 6.6 24/61 39.3 3~ 6.3 31/80 38.8 + 5.4 33182 40.2 f 5.4 25/63 39.7 + 6.2 15/78 19.2 c 4.5 14186 16.3 + 3.9

45

segregating for the pigmentation markers p and cch were analyzed. The prior to sacrifice. Progeny mice were also scored for the presence or absence (see Table 1 and Fig. 1) and the segregation of these loci was followed. The progeny analyzed for each two-point cross; the lower value is the calculated

order that may result from rearrangements of the M, spretus chromosome have so far not been observed (Guenet et al., 1988). The conservation of order suggests the position of the genetic interval on mouse chromosome 7 into which a mouse homolog of myotonic dystrophy (Dym) may map (Fig. 3). Myotonic dystrophy is a human disease locus that has been shown by multipoint genetic mapping in DM families to map to human chromosome 19q in the interval between CKMM and D19S50 (Brunner et al., 1989; Johnson et al., 1989; Korneluk et at., 1989). PRKCG maps distal on human chromosome 19 to D19S50. There is, however, no convincing evidence from biochemical or physiological analyses of a candidate gene product (Renaud, 1987; Harper, 1989). D19S51 is a human CpG island-containing locus derived from a human chromosome 19q/Chinese hamster hybrid, 2OXP3542-1.4 (Stallings et al., 1988). Genetic mapping studies (Johnson et uL, manuscript submitted) have shown that D19S51 maps into the CKMMD19S50 interval and would appear to indicate that D19S51 lies distal to DM. If this is confirmed, this locus would represent a more closely linked distal flanking marker to DM than D19S50 and indicate that DM maps in the small genetic interval between CKMM and this marker. These two markers were mapped in an interspecific backcross and lie some 2.2 + 2.2 CM apart on the proximal portion of mouse chromosome 7. Analysis of other human chromosome 19q markers have failed to show any change in the order of loci in

this mouse syntenic group compared to human chromosome 19q. Malignant hyperthermia sensitivity is a dominant genetic disorder in man and is one of the major causes of death due to anesthesia. The ryanodine receptor gene (Ryr) (Takeshima et al., 1989; Marks et al., 1989) encodes the Ca2+ release channel in the sarcbplasmic reticulum of skeletal muscle cells and can be considered a candidate for the MHS mutation. The MHS locus has been mapped to human chromosome 19q13.2/13.1 by McCarthy et al. (1990). These studies have shown

TABLE

3

Classesof Haplotypes Observed in the domesticus/M. spretua Backcross Parental haplotypes Total number of nonrecombinant haplotypes Recombinant haplotypes Total number of recombinant haplotypes 1. cch-p interval 2. p-Myod interval 3. p-Ryr interval 4. Myod-Ryr interval 5. Ryr-Atpainterval 6. Myod-Atpainterval 7. Ckmm-DlSSBlh interval 8. DlSSBlh-Pkcc interval 9. Double recombinant haplotyes Total

backcross

progeny

analyzed

M.

46 40 12 3 1 11 2 6 1 2 2 86

16

CAVANNA

ET

AL.

MOUSE NUMBER 42

19

32

61

60

24

Pkcc -

D

S

D

D

D

S

D19SSlh’

s

S

D

D

: S

i S

E D

E D

S

D

D

D

S

D

S

D

P /

S

D

S

D

ch c /

S

D

S

S

ChJtIl T&l Atpa-

RYr

Myod

I

/

/

SCM

I

FIG. 2. Pedigree analysis of markers on mouse chromosome 7. Each progeny mouse received a M. domesticus chromosome and the above recombinant chromosome from its parents. DNAs from progeny mice were digested with the appropriate restriction enzyme, hybridized to each probe, and scored for the presence of A4. spretus RFLVs (S). Where no M. spretus RFLV was present, mice were scored as possessing the M. domes&us (D) RFLV. Probe/mouse combinations that were not tested are denoted by-. The above order was deduced by a simple pedigree analysis, utilizing a minimum number of crossovers to define probe order.

that MHS is linked with a maximum lod score of 5.7 at the CYPBA locus in the 20-CM interval between D19S9 and APOCB; CYP2A maps 3 CM proximal to ATPlA3 on human 19q (see Fig. 3). GPI (glucose phosphate isomerase) has been mapped to this interval (Schonk et al., 1989). Analysis of pigs has revealed a porcine homolog (Hal) to this human disorder and the genetics of this porcine syndrome have been extensively studied (Archibald and Imlah, 1985). Hal is closely linked to the glucose phosphate isomerase locus on pig chromosome 6, a locus that also maps to mouse chromosome 7 (Davisson et al., 1989). In mouse Gpi maps some 5 CM distal to Atpa- on chromosome 7 (Davisson et al., 1989; Kent et al., 19871, indicating that the mouse homolog of MHS would map in this region of mouse chromosome 7. The map location of the ryanodine receptor locus in humans or pigs has not been determined. However, the Ryr locus has been mapped in our interspecific backcross and is located some 5 -I- 2 CM distal to Atpa- on mouse chromosome 7. This is in concordance with the

predicted position of the murine homolog of MHS. RYR has not yet been mapped in human families segregating for MHS, but the data in this paper would predict that RYR would map close to the MHS or, indeed, could cosegregate with the defect itself. The comparative maps of human chromosome 19q and mouse chromosome 7 show no differences in gene order but do exhibit variations in genetic distances between some of the loci (see Fig. 3). The PRKCG and D19S51 distance on human chromosome 19 is approximately 25 CM in comparison to 3.6 + 2.5 CM for the distance between Pkcc and D19S51h on mouse chromosome 7. Atpaand Ckmm on mouse chromosome 7 fail to segregate, whereas the same loci on human chromosome 19q span 15 CM. Nevertheless, it is important to note that the human DlSS51-CKMM distance of 2.5 CM is not significantly different from the mouse DlSS5lh-Ckmm distance of 2.2 2 2.2 CM. However, the high density of human chromosome 19q markers on mouse chromosome 7, spanning some 8 CM, compared with the same group on human chro-

MOUSE

CHROMOSOME

7 AND

HUMAN

CHROMOSOME

17

HOMOLOGY

4

tel

/-

- PRKCG

/ / / / / / / / / / / / / MMU7

/

/

/ /

DYm

/

/

,

- CKMM

1

DM

1OcM

- ATPlA3

I/ ,

- D19S51

/

- MHS

CYP2A

tel t

I

HSA 19q FIG. 3. Comparative genetic maps of mouse chromosome 7 and human chromosome 19q. Comparative genetic maps of proximal mouse chromosome 7 (MMU7) and human chromosome 19q (HSAlSq). Human genetic distances, apart from PRKCG-CKMM, were derived from summary genetic maps (male recombination distances) of the Report of the Committee on Linkage and Gene Order (15). For the PRKCGCKMM distance, also derived from (15), the sex is not determined. The figure shows the conservation of order of loci in the syntenic groups on these two chromosomes. Also shown are the large differences in genetic distances between loci on the two chromosomes (see text for further discussion). The two human disease loci myotonic dystrophy (DM) and susceptibility to malignant hyperthermia (MHS) are shown on the human chromosome 19q map along with their putative homologs (Dym and Ryr, respectively) on mouse chromosome 7.

mosome 19q spanning 60 CM, may be indicative of centromeric interference of recombination on mouse chromosome 7. Such centromeric interference has been suggested as the cause of the discrepancy in genetic distances between human and mouse X chromosomes (Disteche et al., 1989).

ACKNOWLEDGMENTS The authors thank Jessica Buxton and Peggy Shelbourne for help with DNA probes. This work was supported by grants to S.D.M.B. (Birthright Grant B3/87) and to K.J.J. (Muscular Dystrophy Group Grant RA3/257/1; Muscular Dystrophy Association). Note added in proof. MacLennan et al. (1990, Nature (London) 343: 559-561) have reported the cosegregation of the ryanodine receptor locus (RYR) with MHS in human families and the localisation of the RYR locus to human chromosome 19q13.1.

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