Proc. Natl. Acad. Sci. USA Vol. 87, pp. 8642-8646, November 1990 Genetics
Molecular mapping of the mouse db mutation (obesity/genetic mapping/diabetes)
N. BAHARY*, R. L. LEIBELt, L. JOSEPH*t, AND J. M. FRIEDMAN*t§ tHoward Hughes Medical Institute, *Laboratory of Molecular Cell Biology, tLaboratory of Human Behavior and Metabolism, Rockefeller University, New York, NY 10021
Communicated by Alexander G. Bearn, May 16, 1990
Diabetes (db) is an autosomal recessive muABSTRACT tation located in the midportion of mouse chromosome 4 that results in profound obesity with hyperphagia, increased metabolic efficiency, and insulin resistance. To clone this gene and generate a molecular map of the region around this mutation, two genetic crosses were established: an intraspecific backcross between C57BL/6J db/db females and C57BL/6J db/db x DBA/2J +/+ F1 (B6D2 db/+ FI) male mice and an interspecific intercross between B6D2 db/ + F1 males and C57BL/6J db/db x Mus spretus F1 (B6spretus db/+ F1) females. The progeny of both crosses were characterized for genotype at the db locus to map a series of restriction fragment length polymorphisms relative to the db locus. Measurements of body weight, body length, and plasma concentrations of glucose and insulin in the animals allowed the assignment of genotype (db/db vs. db/+ or +/+). A total of 132 progeny of the intraspecific cross and 48 db/db progeny of the interspecific cross were typed for individual restriction fragment length polymorphisms to generate a gene order of: centromere-brown
of the defect and provides an approach for cloning genes solely on the basis of chromosomal location (10, 11). The initial phase of the reverse genetic approach involves mapping of a mutant gene relative to chromosomal markers, usually restriction fragment length polymorphisms (RFLPs). Toward this end, both an intraspecific and an interspecific backcross segregating db were established to enable the genetic mapping of the db mutation relative to a series of RFLPs on mouse chromosome 4. To unambiguously assign genotype at the db locus and also to examine the effects of genetic (strain) background on the development of diabetes, plasma concentrations of glucose and insulin were assayed in the backcross and intercross progeny at the time of sacrifice. Variance of these parameters among these progeny can also be used to assess the effect of the differing genetic backgrounds of the progenitor strains on the severity of glucose intolerance resulting from homozygosity for db.
(Mt4)-PAMm3_--Ifa (Inta)-Cjun-db-D4Rpl-Glutl-Mtv-13-
Numerous attempts have been made to identify the primary molecular defect in db/db animals. However, because of the multiple metabolic and physiologic abnormalities seen in db/db animals, the separation of primary gene effects from secondary metabolic alterations has proven difficult. For this reason we have taken the approach of "reverse genetics" to isolate the db gene product (9). This approach requires no knowledge or assumptions concerning the biological nature
Mouse Crosses. The intraspecific and interspecific crosses segregating the db mutation were performed as shown in Fig. 1 a and b. Agouti females carrying the transplanted ovaries of C57BL/6J db/db mice (generously provided by Douglas Coleman of The Jackson Laboratory, who has maintained db congenic stocks on C57BL/6J for seven generations) were mated to both DBA/2J +/ + and Mus spretus males (The Jackson Laboratory) (4). F1 black (indicating their origin from transplanted ovarian tissue) male progeny of the C57BL/6J x DBA/2J cross were backcrossed to the female transplants. In addition, F1 males of a C57BL/6J x M. spretus cross were subsequently mated to F1 female offspring of the C57BL/6J x DBA/2J progenitors. All animals were fed an ad libitum diet of Pico laboratory mouse breeding chow 5058 (Purina), containing 9% (wt/wt) fat, and water. Progeny Analysis. Animals were sacrificed by CO2 asphyxiation between 87 and 186 days of age. After a 15-hr fast each mouse had -1.0 ml of whole blood withdrawn by cardiac puncture into an Eppendorf tube containing 50 ml of 82 ttM EDTA as an anticoagulant. The plasma was decanted and frozen at -80'C for subsequent assay of [insulin] (12) and [glucose] (13). All progeny were scored for sex, weight, and length (nose to anus). DNA Preparation and Analysis. High molecular weight DNA was prepared from kidney, spleen, or liver (14) and Southern blotted to GeneScreenPlus (NEN Research Products, Boston) by following the manufacturer's protocol. Probes. The following probes for detecting the loci indicated were used in these experiments. Lck (lymphocytespecific protein-tyrosine kinase, Lck) was the gift of R. Perlmutter (15), Inta (interferon a, Ifa) was from P. PithaRowe (16), PAMm32 [variable number of tandem repeats (VNTR) probe] was from A. Jeffreys (17), MMTV (Mtv-13)
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: RFLP, restriction fragment length polymorphism; BMI, body mass index. §To whom reprint requests should be addressed.
MATERIALS AND METHODS
Lck. Several of the genes that are linked to db [Cjun, glucose transporter (Glut)) and Lck] map to human chromosome 1p, suggesting that db may be part of a syntenic group between human ip and the distal portion of mouse chromosome 4. In addition, phenotyping of the progeny of these crosses revealed a wide range in plasma concentrations of glucose and insulin among the obese progeny, with some animals developing overt diabetes and others remaining euglycemic. Distributions of age-controlled plasma [glucose] and [insulin] among the intraspeciffic-cross obese progeny were not bimodal, suggesting a role for polygenic differences between the progenitor strains (C57BL/6J and DBA/2J) in the development of overt diabetes.
Diabetes (db) is an autosomal recessive mutation located on mouse chromosome 4 that arose spontaneously in the C57BL/KsJ (BL/Ks) strain (1). At least five separate spontaneous mutations allelic with db-db2J, dbad, db3A, db4A, and db pasteur-have been detected since the initial report (2, 3). Animals homozygous for the db mutation are notably obese as early as 4 weeks of age and often develop glucose intolerance and diabetes, the severity of which depends upon the inbred strain on which the mutant allele has been bred
(4-8).
8642
Genetics:
Bahary et al.
Proc. Natl. Acad. Sci. USA 87 (1990)
a
8643
b B -I|B
D-
D
+Id
'- +
SA
[B
db
db
S
x
db1
cb
+
+
C57BL/6J
C57BL/6J db/db transplant
db/db transplant
B]IB
B]
X
B
~D
-dbt
males
D
B
Cb
B
spretus +/+ males
B-s S
C57BL/6J x DBA/2J db/+ males
C57BL/6J db/db transplant
d1b
+/+
C57BL/6J x spretus db/+ females
X
Bz
D
C57BL/6J x D BA/2J db/+ males
C
BD Cb C
N
S
+
obese obese lean Parentals-J LRecombinantsi
D~ eS
lean
RFLP haplotypes, and phenotypes, of N2 animals resulting from: C57BL/6J db/db X B6D2 F1 db'+
Parental
V:
Hecomoonants
RFLP haplotypes for obese progeny (F2) resulting from: B6D2 F1 db/+ X B6spretus F1 db/+
FIG. 1. Description of the crosses performed. (a) The intraspecific backcross was accomplished by mating C57BL/6J db/db ovarian transplants to DBA/2J males. The F1 male progeny were then backcrossed to the ovarian transplants. (b) In an analogous fashion, C57BL/6J db/db ovarian transplants were mated to M. spretus males. Female F1 offspring were mated to F1 males of the C57BL/6J db/db x DBA/2J +/+ cross, and the obese progeny (F2) were analyzed. In this figure, B, D, and S represent the respective C57BL/6J, DBA/2J, and M. spretus alleles for a particular RFLP. For the C57BL/6J x DBA/2J backcross, recombinants are scored when (i) db/db animals carry the DBA/2J allele at a particular locus or (ii) db/+ animals are homozygous for the C57BL/6J allele. For the C57BL/6J x M. spretus intercross, recombinants are scored when obese (db/db) animals carry any combination of the DBA/2J (if one exists) or M. spretus alleles for a particular RFLP. (c) Photograph of db/db and db/+ animals; 5-month-old male littermates derived from the backcross. The animal on the left is db/+ and weighed 26.7 g. The animal on the right is db/db and weighed 83.7 g.
from B. Callahan (18), Mt4 (brown locus, b) was from I. Jackson (19), Glutl (glucose transporter 1) was from M. Birnbaum (20), D4Rp1 (D4Rpl) was from R. Elliot (21), and Cjun (avian sarcoma virus 17 oncogene homologue, Jun) was from P. Vogt (22). Inserts for each of the DNA probes were radiolabeled using [a-32P]dCTP and random hexamers (23) and purified over a Sephadex G-50 spin column. Hybridizations were performed following the manufacturer's protocol for GeneScreenPlus.
plasma [glucose] or [insulin] that was >3 SDs above the mean of their lean littermates, thus confirming their classification as genotypically db/db. For the 58 db/db N2 animals, the mean BMI was 0.582 g/cm2, the mean [glucose] was 488 mg/dl, and the mean [insulin] was 717.4 microunits/ml. The db genotype of three animals with BMIs between 0.400 and 0.450 g/cm2 was assigned on the basis of their plasma [glucose] and [insulin]. 80
RESULTS Phenotypic Characterization of the N2 Animals. The progeny of the intraspecific backcross between C57BL/6J db/db female ovarian transplants and DBA/2J males (Fig. la) are referred to as N2 animals. The offspring of the interspecific intercross (Fig. lb) between B6D2 F1 db/+ males x B6 spretus F1 db/+ females are referred to as F2 animals. Mapping of loci relative to db requires both the assignment of genotype at the db locus and typing the animal for the inheritance of a particular RFLP. Generally, the phenotypic differentiation between db/db and db/+ in the N2 cross and between db/db and db (or +)/+ in the F2 cross was obvious on physical inspection of the animal (Fig. 1c). Nevertheless, to ensure the unambiguous assignment of db genotype, body weight, body mass index [BMI = weight/(nose-to-anus length)2], plasma [glucose] and plasma [insulin] were measured in each animal. Of these four parameters, only BMI produced a clear separation of the animals into two distinct groups with values that generally were either >0.450 or 0.450 g/cm2 were tentatively designated as db/db, whereas those N2 animals with a BMI < 0.400 g/cm2 were considered to be db/ +. Plasma from the N2 animals was assayed for [glucose] and [insulin]. The mean (± SD) [glucose] of the lean animals was 241 ± 78 mg/dl (n = 76) and the mean (± SD) [insulin] was 102.1 ± 65.1 microunits/ml (n = 76). All N2 animals with a BMI > 0.450 g/cm2 had either a
-
0.8 -
70 -
0.7 -
**.
4-
I
E
60
E 0.6-
o,
50
.)
0.5-
.0 3:
40
t
0.4-
.;t.;
?
30
CO
20
#
0
0
:
0.30.2
0
100 200
'
100
c
.1
200
animal ID
animal ID
3000
** *-
4
-
700
-
4
600 44'
*
x, 2000
500
400 ~E 400 )
O- k
3 10005.
**
,#
300 m
200 100
0
100 200
animal ID
0100a200
animal ID
FIG. 2. Analysis of individual N2 animals. Plasma [insulin], [glucose], weight, and BMI (g/cm2) were measured in all N2 animals. The BMI is comparable to the body mass index in man and is used as an indirect measurement of adiposity. ,uu, Microunits; animal ID, animal identification number.
8644
Genetics:
Bahary et al.
Of concern in these experiments was the possibility that some of the db/db N2 animals might become overtly diabetic, lose weight, and thus be mistakenly classified by BMI ( 0.5). The number of N2 males, according to our weekly census, did not change substantially after weaning, suggesting that the less than expected number of db/db N2 males was likely the result of death before 4 weeks of age. RFLP Mapping in the Intraspecific Cross. Initially, five genomic probes, known to map to the region of chromosome 4 near db, were used to type the backcross (N2) progeny for the inheritance of RFLPs between the C57BL/6J and DBA/2J inbred lines. For probes, Inta, PAMm32, and Lck, a polymorphism was detected with the restriction enzyme Bgl II. An RFLP for the Mtv-13 locus detected by the retroviral MMTV probe was scored with EcoRI. The brown locus was mapped using the Mt4 probe for which an RFLP between C57BL/6J and DBA/2J animals is detectable using Taq I (19). The sizes of informative DBA/2J alleles for each probe are as follows: Lck [3.5 kilobases (kb), 1.7 kb], MMTV (9.0 kb), PAMm32 (6.0 kb), Inta (3.2 kb), and Mt4 (4.9 kb). The probes were separately hybridized to restriction digests of DNA obtained from both the db/db and db/+ progeny of the db backcross and stored for the presence of the DBA/2J allele. All N2 animals were scored for Mt4, PAMm32, Inta, and Lck. Phenotypic Characterization of the F2 Animals. To facilitate mapping of probes for which B x D (C57BL/6J x DBA/2J) RFLPs were not readily identifiable, an interspecific intercross between C57BL/6J db/db and M. spretus was developed (24) (Fig. lb). In this cross, DBA/2J +/+ x C57BL/6J db/db F1 males were mated to C57BL/6J db/db x M. spretus +/+ F1 females, and only obese F2 animals were scored. Genotype at the db locus was tentatively assigned on the basis of BMI and of plasma concentrations of glucose and insulin as described for the N2 cross. For the 85 lean control animals, the mean BMI was 0.319 ± 0.049 g/cm2 (+ SD); the plasma [glucose] was 237 + 93 mg/dl; and the plasma [insulin] was 28.2 + 33.4 microunits/ml. Forty-eight obeseappearing progeny from this cross had a BMI > 0.450 g/cm2, and either a plasma [insulin] or [glucose] that was elevated 3
Proc. Natl. Acad. Sci. USA 87 (1990)
SDs above the mean of lean F2 littermates (as defined by BMI, [glucose], and [insulin]). For these 48 animals, the mean BMI was 0.577 g/cm2, the mean [glucose] was 557 mg/dl, and the mean [insulin] was 252.9 microunits/ml. These 48 progeny were tentatively designated as db/db F2 animals. One obese F2 animal rates special attention in this regard. Animal 337, which was originally designated db/db, had a BMI of 0.459 g/cm2 and an [insulin] of 199.4 microunits/ml. This concentration of insulin is elevated >3 SDs above the lean F2 mean of 8.4 ± 10.8 microunits/ml. Despite these data that suggest that this animal is db/db, RFLP analysis of animal 337 revealed M. spretus alleles for all the probes linked to db (described above). Thus, animal 337 is either a wild-type animal with an unusually high BMI or is doubly recombinant between the flanking markers Cjun and D4Rp1. Since the animal carries the M. spretus allele at all loci tested, inclusion of this animal in the mapping panel does not affect the order of the loci shown in the Fig. 4 but does slightly alter the relative distances between the loci. Of note, progeny of interspecific crosses do occasionally attain weights of >50 g (unpublished observation). The F2 obese mice were typed for the presence of the M. spretus allele with the same probes used in the intraspecific cross (with the exception of MMTV) as well as three additional probes: Cjun, Glutl, and D4Rp1. Cjun was scored using Pst I; D4RpJ and Glut] were followed using Taq I. In addition, F2 obese mice were typed for the presence of the DBA/2J alleles for the probes Inta and Lck, by using the RFLPs described earlier for the typing of the N2 animals. By counting recombinants and minimizing double crossovers among the progeny of these crosses (Fig. 3), the genetic map of the region adjacent to the db locus was refined (Fig. 4). The gene order of these loci was: centromere-brown
(Mt4)-PAMm32-Ifa (Inta)-Cjun-db-D4RpJ-Glutl-Mtv-13Lck. In Fig. 4, the location of the loci mapped among progeny of the intraspecific cross are shown above the line, and those for the interspecific cross are shown below the line. Order was determined among the probes D4Rp1, Inta, and Glutl. However, definitive order could not be determined between probes MMTV and either Glutl or D4Rp1, because these probes were not mapped in the same cross. There was only one double crossover across this interval (Fig. 3a), and this order is 10,000-fold more likely than any other (allowing the order of probe MMTV relative to probes Glutl and D4Rp1 to remain undetermined) when the recombination frequencies between all loci are analyzed with the MAPMAKER program (26). Of note, three of the genes that flank db, Cjun, Glutl, and Lck, all map to human chromosome 1p31-36 (27), suggesting that the human homologue ofdb may reside in this interval. Glucose and Insulin Level in N2 db/db Animals. Values of plasma [glucose] and [insulin] among the db/db N2 animals were examined for possible effects of genetic backgroundincluding sex on the diabetogenicity of the db mutation. The plasma [glucose] and [insulin] of the db/db N2 animals were analyzed in three nonoverlapping age groups (3-4, 4-5, and 5-6 months). Analysis of variance of age and sex (28) demonstrated statistically significant effects of age and sex on [insulin] but not on [glucose] (data not shown). Despite the lower [insulin] in males than females and the decline of [insulin] with age in both sexes, no age or sex effect on [glucose] was seen. Of note in all three age groups, a wide range of plasma [glucose] and [insulin] was evident; animals with [glucose] varying from nondiabetic to frankly diabetic were apparent with a relatively continuous distribution. Among the db/db N2 animals the plasma [glucose] ranged between 218 and 698 mg/dl and the plasma [insulin] ranged between 106.2 and 2160.8 microunits/ml. There was no evidence of a bimodal distribution of plasma [glucose] or [insulin] among the db/db backcross progeny (Fig. 2); i.e.,
Genetics:
Bahary et al.
Proc. Natl. Acad. Sci. USA 87 (1990) 1/48
7/128 5.5 (2.0)
MM3
1.7 (1.2)
7/124 5.6 (2.1)
Inta
6/129
4.7(1.9)
12/232 9.1 (2.5)
db
11/132 8.3 (2.4)
81/1 32
2/121
a 8/132 6.0 (2.1)
9/132 6.8 (2.2) MMTV
4/125 3.2 (1.6) 10/132 7.6 (2.3)
17/132 19/125 20/129 12.8 (2.9) 15.2 (3.2) 15.5 (3.2) db
Mt4 MlMl*EJO PX Mm3 .0 Inta db * z MMTV * ]* Lck *5 E 5* ** 146 3 4 3 0 2 db/db N2 animals
EM E
Inta
0 0 0 U 0
0I
2.0 (2.0) 0/48 0.0 (0)
b
29/132
db
3/48 6.2 (3.4)
4/48 8.3 (3.9)
D4RP1
1/48 .2.1 (2.1)
4/48 8.3 (4.0)
4/48 8.3 (4.0)
5/48 10.4 (4.4)
Gt
4/48 8.3 (4.0)
5/48 10.4 (4.4)
11.4 (3.4)
8/48
12/96
Gt
D4RP1
db
Cjun
Inta
Lck
121.9 (3.6)
(2.8)
Cjun
3/48 6.2 (3.4)
MMTV
8.3 (2.4)
4/96 4.1
11/96
16.36(5.4) 12.5 (3.9)
8645
Lck
Mt4
MM3
Inta
cjun
000e00 000z000 000000
0
005000
0
D4RP1
flllOO MElfM[] MHO *
END MEN
GtI Lck
155 6 4 1 2 5
ESOOHE 139 4 3 1 1 01 inheritance of the mus spretus allele
db/+ N2 animals
143 2 3 i inheritance of the DBA/2J allele
FIG. 3. Summary of the results from the intraspecific (a) and interspecific (b) crosses. The approximate map distances in centimorgans [map distance in cM = (no. recombinants/total no. of meioses)*100 between probes was calculated using the program "Spretus Madness" (25); values in parentheses are SD]. Each of the loci mapped in the crosses is listed on the left of the grid; each column represents a possible configuration of inherited DBA/2J and/or M. spretus alleles. The number underneath each column is the number of animals that have inherited that particular pattern of alleles. Solid boxes represent the C57BL/6J alleles and open boxes represent the DBA/2J or M. spretus allele. Gt, (Glutl); Inta (Ifa). numerous animals with [insulin] were notable.
intermediate levels of [glucose] and
Cjun, 5cM proximal to D4RpJ and Mtv-13 (MMTV), 7 cM proximal to Gluti, and 12 cM proximal to Lck (see Fig. 4). This gene order is consistent with that reported (25, 27, 29). Specifically, our data position Mtv-13 distal, and Ifa proximal, to db, as suggested by Huppi et al. (29). Because some probes could not be mapped on both crosses, the order of Mtv-13 relative to Glutl and D4Rpl remains ambiguous. These data demonstrate that db is flanked by a series of RFLPs that have been mapped to human chromosome lp31-36 (27) and suggest that, if there is a human homologue of db, it would probably map to chromosome ip. This information may be useful in the analysis of the heritability of human obesity and type II diabetes. Humans with a defect in the human homologue of the db gene (if one exists) might be expected to cosegregate RFLPs from this chromosome with an obese and/or diabetic phenotype in human pedigrees. Experiments to test this possibility are under way. The characterization of the progeny of the crosses described will permit fine genetic mapping of this region, ultimately narrowing the 95% confidence interval around db to
Molecular mapping of the mouse db mutation (obesity/genetic mapping/diabetes)
N. BAHARY*, R. L. LEIBELt, L. JOSEPH*t, AND J. M. FRIEDMAN*t§ tHoward Hughes Medical Institute, *Laboratory of Molecular Cell Biology, tLaboratory of Human Behavior and Metabolism, Rockefeller University, New York, NY 10021
Communicated by Alexander G. Bearn, May 16, 1990
Diabetes (db) is an autosomal recessive muABSTRACT tation located in the midportion of mouse chromosome 4 that results in profound obesity with hyperphagia, increased metabolic efficiency, and insulin resistance. To clone this gene and generate a molecular map of the region around this mutation, two genetic crosses were established: an intraspecific backcross between C57BL/6J db/db females and C57BL/6J db/db x DBA/2J +/+ F1 (B6D2 db/+ FI) male mice and an interspecific intercross between B6D2 db/ + F1 males and C57BL/6J db/db x Mus spretus F1 (B6spretus db/+ F1) females. The progeny of both crosses were characterized for genotype at the db locus to map a series of restriction fragment length polymorphisms relative to the db locus. Measurements of body weight, body length, and plasma concentrations of glucose and insulin in the animals allowed the assignment of genotype (db/db vs. db/+ or +/+). A total of 132 progeny of the intraspecific cross and 48 db/db progeny of the interspecific cross were typed for individual restriction fragment length polymorphisms to generate a gene order of: centromere-brown
of the defect and provides an approach for cloning genes solely on the basis of chromosomal location (10, 11). The initial phase of the reverse genetic approach involves mapping of a mutant gene relative to chromosomal markers, usually restriction fragment length polymorphisms (RFLPs). Toward this end, both an intraspecific and an interspecific backcross segregating db were established to enable the genetic mapping of the db mutation relative to a series of RFLPs on mouse chromosome 4. To unambiguously assign genotype at the db locus and also to examine the effects of genetic (strain) background on the development of diabetes, plasma concentrations of glucose and insulin were assayed in the backcross and intercross progeny at the time of sacrifice. Variance of these parameters among these progeny can also be used to assess the effect of the differing genetic backgrounds of the progenitor strains on the severity of glucose intolerance resulting from homozygosity for db.
(Mt4)-PAMm3_--Ifa (Inta)-Cjun-db-D4Rpl-Glutl-Mtv-13-
Numerous attempts have been made to identify the primary molecular defect in db/db animals. However, because of the multiple metabolic and physiologic abnormalities seen in db/db animals, the separation of primary gene effects from secondary metabolic alterations has proven difficult. For this reason we have taken the approach of "reverse genetics" to isolate the db gene product (9). This approach requires no knowledge or assumptions concerning the biological nature
Mouse Crosses. The intraspecific and interspecific crosses segregating the db mutation were performed as shown in Fig. 1 a and b. Agouti females carrying the transplanted ovaries of C57BL/6J db/db mice (generously provided by Douglas Coleman of The Jackson Laboratory, who has maintained db congenic stocks on C57BL/6J for seven generations) were mated to both DBA/2J +/ + and Mus spretus males (The Jackson Laboratory) (4). F1 black (indicating their origin from transplanted ovarian tissue) male progeny of the C57BL/6J x DBA/2J cross were backcrossed to the female transplants. In addition, F1 males of a C57BL/6J x M. spretus cross were subsequently mated to F1 female offspring of the C57BL/6J x DBA/2J progenitors. All animals were fed an ad libitum diet of Pico laboratory mouse breeding chow 5058 (Purina), containing 9% (wt/wt) fat, and water. Progeny Analysis. Animals were sacrificed by CO2 asphyxiation between 87 and 186 days of age. After a 15-hr fast each mouse had -1.0 ml of whole blood withdrawn by cardiac puncture into an Eppendorf tube containing 50 ml of 82 ttM EDTA as an anticoagulant. The plasma was decanted and frozen at -80'C for subsequent assay of [insulin] (12) and [glucose] (13). All progeny were scored for sex, weight, and length (nose to anus). DNA Preparation and Analysis. High molecular weight DNA was prepared from kidney, spleen, or liver (14) and Southern blotted to GeneScreenPlus (NEN Research Products, Boston) by following the manufacturer's protocol. Probes. The following probes for detecting the loci indicated were used in these experiments. Lck (lymphocytespecific protein-tyrosine kinase, Lck) was the gift of R. Perlmutter (15), Inta (interferon a, Ifa) was from P. PithaRowe (16), PAMm32 [variable number of tandem repeats (VNTR) probe] was from A. Jeffreys (17), MMTV (Mtv-13)
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: RFLP, restriction fragment length polymorphism; BMI, body mass index. §To whom reprint requests should be addressed.
MATERIALS AND METHODS
Lck. Several of the genes that are linked to db [Cjun, glucose transporter (Glut)) and Lck] map to human chromosome 1p, suggesting that db may be part of a syntenic group between human ip and the distal portion of mouse chromosome 4. In addition, phenotyping of the progeny of these crosses revealed a wide range in plasma concentrations of glucose and insulin among the obese progeny, with some animals developing overt diabetes and others remaining euglycemic. Distributions of age-controlled plasma [glucose] and [insulin] among the intraspeciffic-cross obese progeny were not bimodal, suggesting a role for polygenic differences between the progenitor strains (C57BL/6J and DBA/2J) in the development of overt diabetes.
Diabetes (db) is an autosomal recessive mutation located on mouse chromosome 4 that arose spontaneously in the C57BL/KsJ (BL/Ks) strain (1). At least five separate spontaneous mutations allelic with db-db2J, dbad, db3A, db4A, and db pasteur-have been detected since the initial report (2, 3). Animals homozygous for the db mutation are notably obese as early as 4 weeks of age and often develop glucose intolerance and diabetes, the severity of which depends upon the inbred strain on which the mutant allele has been bred
(4-8).
8642
Genetics:
Bahary et al.
Proc. Natl. Acad. Sci. USA 87 (1990)
a
8643
b B -I|B
D-
D
+Id
'- +
SA
[B
db
db
S
x
db1
cb
+
+
C57BL/6J
C57BL/6J db/db transplant
db/db transplant
B]IB
B]
X
B
~D
-dbt
males
D
B
Cb
B
spretus +/+ males
B-s S
C57BL/6J x DBA/2J db/+ males
C57BL/6J db/db transplant
d1b
+/+
C57BL/6J x spretus db/+ females
X
Bz
D
C57BL/6J x D BA/2J db/+ males
C
BD Cb C
N
S
+
obese obese lean Parentals-J LRecombinantsi
D~ eS
lean
RFLP haplotypes, and phenotypes, of N2 animals resulting from: C57BL/6J db/db X B6D2 F1 db'+
Parental
V:
Hecomoonants
RFLP haplotypes for obese progeny (F2) resulting from: B6D2 F1 db/+ X B6spretus F1 db/+
FIG. 1. Description of the crosses performed. (a) The intraspecific backcross was accomplished by mating C57BL/6J db/db ovarian transplants to DBA/2J males. The F1 male progeny were then backcrossed to the ovarian transplants. (b) In an analogous fashion, C57BL/6J db/db ovarian transplants were mated to M. spretus males. Female F1 offspring were mated to F1 males of the C57BL/6J db/db x DBA/2J +/+ cross, and the obese progeny (F2) were analyzed. In this figure, B, D, and S represent the respective C57BL/6J, DBA/2J, and M. spretus alleles for a particular RFLP. For the C57BL/6J x DBA/2J backcross, recombinants are scored when (i) db/db animals carry the DBA/2J allele at a particular locus or (ii) db/+ animals are homozygous for the C57BL/6J allele. For the C57BL/6J x M. spretus intercross, recombinants are scored when obese (db/db) animals carry any combination of the DBA/2J (if one exists) or M. spretus alleles for a particular RFLP. (c) Photograph of db/db and db/+ animals; 5-month-old male littermates derived from the backcross. The animal on the left is db/+ and weighed 26.7 g. The animal on the right is db/db and weighed 83.7 g.
from B. Callahan (18), Mt4 (brown locus, b) was from I. Jackson (19), Glutl (glucose transporter 1) was from M. Birnbaum (20), D4Rp1 (D4Rpl) was from R. Elliot (21), and Cjun (avian sarcoma virus 17 oncogene homologue, Jun) was from P. Vogt (22). Inserts for each of the DNA probes were radiolabeled using [a-32P]dCTP and random hexamers (23) and purified over a Sephadex G-50 spin column. Hybridizations were performed following the manufacturer's protocol for GeneScreenPlus.
plasma [glucose] or [insulin] that was >3 SDs above the mean of their lean littermates, thus confirming their classification as genotypically db/db. For the 58 db/db N2 animals, the mean BMI was 0.582 g/cm2, the mean [glucose] was 488 mg/dl, and the mean [insulin] was 717.4 microunits/ml. The db genotype of three animals with BMIs between 0.400 and 0.450 g/cm2 was assigned on the basis of their plasma [glucose] and [insulin]. 80
RESULTS Phenotypic Characterization of the N2 Animals. The progeny of the intraspecific backcross between C57BL/6J db/db female ovarian transplants and DBA/2J males (Fig. la) are referred to as N2 animals. The offspring of the interspecific intercross (Fig. lb) between B6D2 F1 db/+ males x B6 spretus F1 db/+ females are referred to as F2 animals. Mapping of loci relative to db requires both the assignment of genotype at the db locus and typing the animal for the inheritance of a particular RFLP. Generally, the phenotypic differentiation between db/db and db/+ in the N2 cross and between db/db and db (or +)/+ in the F2 cross was obvious on physical inspection of the animal (Fig. 1c). Nevertheless, to ensure the unambiguous assignment of db genotype, body weight, body mass index [BMI = weight/(nose-to-anus length)2], plasma [glucose] and plasma [insulin] were measured in each animal. Of these four parameters, only BMI produced a clear separation of the animals into two distinct groups with values that generally were either >0.450 or 0.450 g/cm2 were tentatively designated as db/db, whereas those N2 animals with a BMI < 0.400 g/cm2 were considered to be db/ +. Plasma from the N2 animals was assayed for [glucose] and [insulin]. The mean (± SD) [glucose] of the lean animals was 241 ± 78 mg/dl (n = 76) and the mean (± SD) [insulin] was 102.1 ± 65.1 microunits/ml (n = 76). All N2 animals with a BMI > 0.450 g/cm2 had either a
-
0.8 -
70 -
0.7 -
**.
4-
I
E
60
E 0.6-
o,
50
.)
0.5-
.0 3:
40
t
0.4-
.;t.;
?
30
CO
20
#
0
0
:
0.30.2
0
100 200
'
100
c
.1
200
animal ID
animal ID
3000
** *-
4
-
700
-
4
600 44'
*
x, 2000
500
400 ~E 400 )
O- k
3 10005.
**
,#
300 m
200 100
0
100 200
animal ID
0100a200
animal ID
FIG. 2. Analysis of individual N2 animals. Plasma [insulin], [glucose], weight, and BMI (g/cm2) were measured in all N2 animals. The BMI is comparable to the body mass index in man and is used as an indirect measurement of adiposity. ,uu, Microunits; animal ID, animal identification number.
8644
Genetics:
Bahary et al.
Of concern in these experiments was the possibility that some of the db/db N2 animals might become overtly diabetic, lose weight, and thus be mistakenly classified by BMI ( 0.5). The number of N2 males, according to our weekly census, did not change substantially after weaning, suggesting that the less than expected number of db/db N2 males was likely the result of death before 4 weeks of age. RFLP Mapping in the Intraspecific Cross. Initially, five genomic probes, known to map to the region of chromosome 4 near db, were used to type the backcross (N2) progeny for the inheritance of RFLPs between the C57BL/6J and DBA/2J inbred lines. For probes, Inta, PAMm32, and Lck, a polymorphism was detected with the restriction enzyme Bgl II. An RFLP for the Mtv-13 locus detected by the retroviral MMTV probe was scored with EcoRI. The brown locus was mapped using the Mt4 probe for which an RFLP between C57BL/6J and DBA/2J animals is detectable using Taq I (19). The sizes of informative DBA/2J alleles for each probe are as follows: Lck [3.5 kilobases (kb), 1.7 kb], MMTV (9.0 kb), PAMm32 (6.0 kb), Inta (3.2 kb), and Mt4 (4.9 kb). The probes were separately hybridized to restriction digests of DNA obtained from both the db/db and db/+ progeny of the db backcross and stored for the presence of the DBA/2J allele. All N2 animals were scored for Mt4, PAMm32, Inta, and Lck. Phenotypic Characterization of the F2 Animals. To facilitate mapping of probes for which B x D (C57BL/6J x DBA/2J) RFLPs were not readily identifiable, an interspecific intercross between C57BL/6J db/db and M. spretus was developed (24) (Fig. lb). In this cross, DBA/2J +/+ x C57BL/6J db/db F1 males were mated to C57BL/6J db/db x M. spretus +/+ F1 females, and only obese F2 animals were scored. Genotype at the db locus was tentatively assigned on the basis of BMI and of plasma concentrations of glucose and insulin as described for the N2 cross. For the 85 lean control animals, the mean BMI was 0.319 ± 0.049 g/cm2 (+ SD); the plasma [glucose] was 237 + 93 mg/dl; and the plasma [insulin] was 28.2 + 33.4 microunits/ml. Forty-eight obeseappearing progeny from this cross had a BMI > 0.450 g/cm2, and either a plasma [insulin] or [glucose] that was elevated 3
Proc. Natl. Acad. Sci. USA 87 (1990)
SDs above the mean of lean F2 littermates (as defined by BMI, [glucose], and [insulin]). For these 48 animals, the mean BMI was 0.577 g/cm2, the mean [glucose] was 557 mg/dl, and the mean [insulin] was 252.9 microunits/ml. These 48 progeny were tentatively designated as db/db F2 animals. One obese F2 animal rates special attention in this regard. Animal 337, which was originally designated db/db, had a BMI of 0.459 g/cm2 and an [insulin] of 199.4 microunits/ml. This concentration of insulin is elevated >3 SDs above the lean F2 mean of 8.4 ± 10.8 microunits/ml. Despite these data that suggest that this animal is db/db, RFLP analysis of animal 337 revealed M. spretus alleles for all the probes linked to db (described above). Thus, animal 337 is either a wild-type animal with an unusually high BMI or is doubly recombinant between the flanking markers Cjun and D4Rp1. Since the animal carries the M. spretus allele at all loci tested, inclusion of this animal in the mapping panel does not affect the order of the loci shown in the Fig. 4 but does slightly alter the relative distances between the loci. Of note, progeny of interspecific crosses do occasionally attain weights of >50 g (unpublished observation). The F2 obese mice were typed for the presence of the M. spretus allele with the same probes used in the intraspecific cross (with the exception of MMTV) as well as three additional probes: Cjun, Glutl, and D4Rp1. Cjun was scored using Pst I; D4RpJ and Glut] were followed using Taq I. In addition, F2 obese mice were typed for the presence of the DBA/2J alleles for the probes Inta and Lck, by using the RFLPs described earlier for the typing of the N2 animals. By counting recombinants and minimizing double crossovers among the progeny of these crosses (Fig. 3), the genetic map of the region adjacent to the db locus was refined (Fig. 4). The gene order of these loci was: centromere-brown
(Mt4)-PAMm32-Ifa (Inta)-Cjun-db-D4RpJ-Glutl-Mtv-13Lck. In Fig. 4, the location of the loci mapped among progeny of the intraspecific cross are shown above the line, and those for the interspecific cross are shown below the line. Order was determined among the probes D4Rp1, Inta, and Glutl. However, definitive order could not be determined between probes MMTV and either Glutl or D4Rp1, because these probes were not mapped in the same cross. There was only one double crossover across this interval (Fig. 3a), and this order is 10,000-fold more likely than any other (allowing the order of probe MMTV relative to probes Glutl and D4Rp1 to remain undetermined) when the recombination frequencies between all loci are analyzed with the MAPMAKER program (26). Of note, three of the genes that flank db, Cjun, Glutl, and Lck, all map to human chromosome 1p31-36 (27), suggesting that the human homologue ofdb may reside in this interval. Glucose and Insulin Level in N2 db/db Animals. Values of plasma [glucose] and [insulin] among the db/db N2 animals were examined for possible effects of genetic backgroundincluding sex on the diabetogenicity of the db mutation. The plasma [glucose] and [insulin] of the db/db N2 animals were analyzed in three nonoverlapping age groups (3-4, 4-5, and 5-6 months). Analysis of variance of age and sex (28) demonstrated statistically significant effects of age and sex on [insulin] but not on [glucose] (data not shown). Despite the lower [insulin] in males than females and the decline of [insulin] with age in both sexes, no age or sex effect on [glucose] was seen. Of note in all three age groups, a wide range of plasma [glucose] and [insulin] was evident; animals with [glucose] varying from nondiabetic to frankly diabetic were apparent with a relatively continuous distribution. Among the db/db N2 animals the plasma [glucose] ranged between 218 and 698 mg/dl and the plasma [insulin] ranged between 106.2 and 2160.8 microunits/ml. There was no evidence of a bimodal distribution of plasma [glucose] or [insulin] among the db/db backcross progeny (Fig. 2); i.e.,
Genetics:
Bahary et al.
Proc. Natl. Acad. Sci. USA 87 (1990) 1/48
7/128 5.5 (2.0)
MM3
1.7 (1.2)
7/124 5.6 (2.1)
Inta
6/129
4.7(1.9)
12/232 9.1 (2.5)
db
11/132 8.3 (2.4)
81/1 32
2/121
a 8/132 6.0 (2.1)
9/132 6.8 (2.2) MMTV
4/125 3.2 (1.6) 10/132 7.6 (2.3)
17/132 19/125 20/129 12.8 (2.9) 15.2 (3.2) 15.5 (3.2) db
Mt4 MlMl*EJO PX Mm3 .0 Inta db * z MMTV * ]* Lck *5 E 5* ** 146 3 4 3 0 2 db/db N2 animals
EM E
Inta
0 0 0 U 0
0I
2.0 (2.0) 0/48 0.0 (0)
b
29/132
db
3/48 6.2 (3.4)
4/48 8.3 (3.9)
D4RP1
1/48 .2.1 (2.1)
4/48 8.3 (4.0)
4/48 8.3 (4.0)
5/48 10.4 (4.4)
Gt
4/48 8.3 (4.0)
5/48 10.4 (4.4)
11.4 (3.4)
8/48
12/96
Gt
D4RP1
db
Cjun
Inta
Lck
121.9 (3.6)
(2.8)
Cjun
3/48 6.2 (3.4)
MMTV
8.3 (2.4)
4/96 4.1
11/96
16.36(5.4) 12.5 (3.9)
8645
Lck
Mt4
MM3
Inta
cjun
000e00 000z000 000000
0
005000
0
D4RP1
flllOO MElfM[] MHO *
END MEN
GtI Lck
155 6 4 1 2 5
ESOOHE 139 4 3 1 1 01 inheritance of the mus spretus allele
db/+ N2 animals
143 2 3 i inheritance of the DBA/2J allele
FIG. 3. Summary of the results from the intraspecific (a) and interspecific (b) crosses. The approximate map distances in centimorgans [map distance in cM = (no. recombinants/total no. of meioses)*100 between probes was calculated using the program "Spretus Madness" (25); values in parentheses are SD]. Each of the loci mapped in the crosses is listed on the left of the grid; each column represents a possible configuration of inherited DBA/2J and/or M. spretus alleles. The number underneath each column is the number of animals that have inherited that particular pattern of alleles. Solid boxes represent the C57BL/6J alleles and open boxes represent the DBA/2J or M. spretus allele. Gt, (Glutl); Inta (Ifa). numerous animals with [insulin] were notable.
intermediate levels of [glucose] and
Cjun, 5cM proximal to D4RpJ and Mtv-13 (MMTV), 7 cM proximal to Gluti, and 12 cM proximal to Lck (see Fig. 4). This gene order is consistent with that reported (25, 27, 29). Specifically, our data position Mtv-13 distal, and Ifa proximal, to db, as suggested by Huppi et al. (29). Because some probes could not be mapped on both crosses, the order of Mtv-13 relative to Glutl and D4Rpl remains ambiguous. These data demonstrate that db is flanked by a series of RFLPs that have been mapped to human chromosome lp31-36 (27) and suggest that, if there is a human homologue of db, it would probably map to chromosome ip. This information may be useful in the analysis of the heritability of human obesity and type II diabetes. Humans with a defect in the human homologue of the db gene (if one exists) might be expected to cosegregate RFLPs from this chromosome with an obese and/or diabetic phenotype in human pedigrees. Experiments to test this possibility are under way. The characterization of the progeny of the crosses described will permit fine genetic mapping of this region, ultimately narrowing the 95% confidence interval around db to
