Genetics: Published Articles Ahead of Print, published on September 30, 2004 as 10.1534/genetics.104.034033
Mapping a syntenic modifier Mapping a syntenic modifier on mouse chromosome 1 influencing the expressivity of the Compact phenotype in the myostatin mutant (MstnCmpt-dl1Abc) Compact mouse
László Varga*, Orsolya Pinke*, Géza Müller†, Balázs Kovács*, Edit Korom*, Gyula Szabó* and Morris Soller‡
* Institute for Animal Biology, Agricultural Biotechnology Center, H-2101 Gödöllő, Hungary † Egis Pharmaceuticals Ltd., H-1475 Budapest, Hungary ‡ Department of Genetics, The Alexander Silberman Life Science Institute, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel
1
Mapping a syntenic modifier Running head: Mapping a syntenic modifier
Keywords: myostatin, hypermuscularity, syntenic, modifier, mapping,
Corresponding author: Dr. László Varga, Institute for Animal Biology, Agricultural Biotechnology Center, P.O.B. 411, H-2101 Gödöllő, Hungary. E-mail:
[email protected]
2
Mapping a syntenic modifier ABSTRACT A novel method to map a modifer gene that is syntenic to its major gene was used to map a male-sex-limited modifier of the expressivity of the Compact phenotype in the myostatin mutant (MstnCmpt-dl1Abc) Compact mouse. The modifier was mapped to the general region of D1Mit262, 40 cM distal to Mstn on chromosome 1. Myogenin, a postulated downstream target of myostatin, maps to the same region.
Genetic analysis of the Hungarian subpopulation of the hypermuscular Compact mouse uncovered a major gene (Cmpt) in the proximal region of mouse chromosome 1, and indicated that additional modifiers are involved in determining the full expressivity of the Compact phenotype (VARGA et al. 1997). A very similar phenotype appeared in mice when the myostatin gene (Mstn), a member of the TGF-β family, was disrupted by gene targeting (MCPHERRON and LEE 1997). Mutations causing hypermuscularity in the double-muscled Belgian Blue and Piedmontese cattle breeds were also found in the myostatin gene (KAMBADUR et al. 1997), and the presence of modifier genes was also indicated in these spontaneous mutants (GROBET et al. 1997; MCPHERRON and LEE 1997). The first mutation in the human myostatin gene was identified recently, at which transition at the splice donor site in intron 1 results in a severely truncated protein causing hypermuscularity in the affected child (SCHUELKE et al. 2004). Sequencing of the myostatin gene of the Compact mouse mutant revealed a 12 bp deletion denoted MstnCmpt-dl1Abc in the propeptid region of the myostatin precursor. The deletion mutant segregated together with the Compact trait, and can be considered as the causative mutation responsible for the hypermuscular phenotype (SZABÓ et al. 1998). The activity of mature myostatin is not zero in homozygous Cmpt/Cmpt mutants (SZABÓ et al. 1998), enabling genes modifying the expression of Mstn, or modulating downstream signaling to have a significant influence on the hypermuscular phenotype. 3
Mapping a syntenic modifier To map modifier loci affecting expressivity of the Compact phenotype, an interspecific mapping population consisting of 2373 F2 progeny was generated by a cross between Comp9, an inbred line homozygous for MstnCmpt-dl1Abc and CAST/Ei, an inbred line derived from Mus musculus castaneus, carrying the wild type allele at Mstn. On the hypothesis that the expressivity of the Compact phenotype of MstnCmpt-dl1Abc is modulated by modifier genes, the allele frequencies of particular modifier alleles in the F2 population is expected to differ between homozygous MstnCmpt-dl1Abc animals with low compact score and those with high compact score. Based on gender, phenotypic and genotypic data, the VARGA et al. (2003) study defined four gender/phenotype/genotype (GPG) groups for analysis all having homozygous CK genotype at Mstn: M1CKCK, M5CKCK, F1CKCK, F4CKCK (see Table 1 for notation, and Table 2 for number of animals in each GPG group. For greater clarity, notation is changed somewhat from VARGA et al. (2003)). By comparing marker allele frequencies in these GPG groups, significant effects on Compact trait expression were found for markers on chromosomes 3, 5, 7, 11, 16 and X (VARGA et al. 2003); the modifier loci apparently exerted their effects on muscularity only in the presence of MstnCmpt-dl1Abc. All F2 mice in the above analysis carried a uniform Comp9 genotype at the Mstn locus, while for markers progressively more distant from Mstn, the Comp9 marker genotype is gradually converted to include heterozygous and homozygous CAST/Ei marker genotypes. Since the large overall excess of Comp9-genotype chromosome 1 alleles in the F2, would confound the analysis for modifier effects on chromosome 1, this chromosome was not included in the original scan for modifiers. In the present study, a statistical method was developed that enabled chromosome 1 to be examined for syntenic modifier loci affecting Compact trait expression. The method is based on marker allele frequencies in the above four F2 progeny groups, and in two additional “control” groups having
4
Mapping a syntenic modifier homozygous CB genotype at Mstn: M1CBCB and F1CBCB. The argument underlying the proposed method is as follows: On the basis of previous studies (VARGA et al. 2003) it can be assumed that parental line genotypes were: Comp9: CKSiKDK/CKSiKDK; CAST/Ei: CBSiBDB/CBSiBDB. The F2 population was generated by crossing the parental lines, and intercrossing the F1 progeny. The F2 individuals were sexed, phenotyped, and genotyped for Mstn; the six GPG groups described above were formed and genotyped for all markers, Si. By elementary genetics, among F2 chromosomes carrying allele CK, there will be (1-rD) having haplotype CKDK, rD having haplotype CKDB, (1-rSi) having haplotype CKSiK, and rSi having haplotype CKSiB. Thus, among the CKCK individuals of the F2, there will be DKDK, DKDB, DBDB individuals in proportion (1-rD)2, 2rD(1-rD), and rD2, respectively; and SiKSiK, SiKSiB, SiBSiB individuals in proportion (1rSi)2, 2(1-rSi)rSi, and rSi2, respectively. The M1CKCK, M5CKCK, F1CKCK, F4CKCK GPG groups, however, will be under selection for the modifier, D. In particular, there will be selection for DK alleles (and against DB alleles) in the M5CKCK and F4CKCK groups, and selection for DB alleles (and against DK alleles) in the M1CKCK and F1CKCK groups. Thus, frequency of DK and DB alleles will differ among the GPGs: the frequency of DK will be higher in the M5CKCK and F4CKCK groups, and lower in the M1CKCK and F1CKCK groups; the opposite will hold for the DB alleles. The change in frequency of DK as a result of selection for high or low compact score will equal (FALCONER 1960): f(D) = IDpKqB where, pK = frequency of allele DK = (1-rD) qB = frequency of allele DB = rD 5
Mapping a syntenic modifier ID = the standardized intensity of selection on DK. This will be a function of the proportion selected, and the effects of DK and DB relative to the phenotypic standard deviation of the trait. For our purposes, the exact value of ID is immaterial. Because the DK and DB alleles are in coupling linkage with SiK and SiB marker alleles, respectively, selection on DK and DB will have a hitchhiker effect (i.e., will exert indirect selection) for SiK and SiB alleles. This will cause marker allele frequencies in the GPG groups to differ from expected. The intensity of selection, ISi, exerted by D on allele frequencies at marker Si will be directly proportional to (1-rDSi). That is, the smaller the proportion of recombination between the marker locus and the modifier locus, the greater the indirect selection on the marker locus. In particular, ISi = ID(1-rDSi) Consequently, the change in allele frequency at the marker locus, will be f(Si) = ISipiqi Thus, finding a significant difference (e.g., by Chi-square contingency test) in marker allele frequency between the M1CKCK and M5CKCK GPGs, and between the F1CKCK and F4CKCK GPGs is indicative of the presence of a modifier locus. The modifier will be located nearest to the marker locus for which ISi is greatest. From the expressions above, we have ISi = f(Si)/piqi. where, pi and qi can be estimated by the consensus map distance of Si from Mstn. However, since the present F2 is an interspecific cross, which may have different map distances than the consensus cross, it is preferable to estimate map distance from the F2 itself. Since the modifiers do not have an effect independent of the homozygous CKCK genotype (VARGA et al. 2003), this can be done by calculating the proportion of recombination between Mstn and Si in the M1CBCB and F1CBCB control groups. 6
Mapping a syntenic modifier On this basis, f(Si) for males is estimated as the difference in pi between the M5CKCK and (M1CBCB+F1CBCB) groups; for females, it is estimated as the difference in pi between the F4CKCK and (M1CBCB+F1CBCB) GPG groups. The distribution of marker genotypes for the M1CBCB and F1CBCB groups across all markers did not differ significantly. Thus, the two genders were combined (Table 2). There was a highly significant difference in the distribution of marker genotypes between the M1CKCK and M5CKCK GPG groups, as expected in the presence of a modifier gene (Table 3). The difference was primarily due to the markers D1Mit309, D1Mit262, and D1Mit33. In contrast, the distribution of marker genotypes for F1CKCK and F4CKCK did not differ significantly. This shows that the modifier gene did not affect Compact trait expression in the female. Thus, the distributions of the F1CKCK and F4CKCK groups were combined, to provide an independent estimate of the distribution of marker genotypes in the absence of the modifier effect (Table 2). The distributions of the combined (M1+F1)CBCB and (F1+F4)CKCK groups were kept separate, so as to provide two independent comparisons for the M1CKCK and M5CKCK groups. The distribution of marker genotypes in the M5CKCK group differed significantly from the distribution of the combined (M1+F1)CBCB group and also from the distribution of the combined (F1+F4)CKCK group (Table 3). Again, the difference was primarily due to the markers in the region D1Mit309 to D1Mit33. In contrast, the distribution of marker genotypes in the M1CKCK group did not differ significantly from the two control distributions. This will be considered in more detail in the following section. Table 4 shows the proportion of recombination between marker and Mstn , for the two control groups (M1+F1)CBCB and (F1+F4)CKCK, and for the M1CKCK and M5CKCK “experimental” groups. The proportion of recombination shown by the (M1+F1)CBCB control was generally somewhat greater than that expected on the basis of map distance; that shown by the (F1+F4)CKCK was generally very 7
Mapping a syntenic modifier similar to the expected. Among the experimental groups, the M1CKCK group showed a higher proportion of recombination across the entire region D1Mit10 to D1Mit37; the M5CKCK group showed a lower proportion of recombination across this region. Both of these effects are as expected on the theory developed in methods, and suggests that a male-sex-limited modifier locus is located in this region. The reason for the lack of significance of the M1CKCK group compared to the controls is now evident. The proportion of recombination for the controls in the region D1Mit262 to D1Mit37 is high. Hence, a further increase is difficult to achieve due to double crossing over. In contrast, a decrease, as expected for the M5CKCK group would be readily apparent. Table 4 also shows values of ISi calculated from the difference in proportion of recombination between the controls and the two experimental groups (M1CKCK and M5CKCK). ISi was calculated separately for each of the control groups. There is a clear maximum in the region DiMit309 to D1Mit33, which suggests that the modifier is located close to the center of the region, at D1Mit262. Although the map distance between these markers and Mstn approaches 0.5 M, the expected proportion of recombination between Mstn and these markers is in the range 0.25 to 0.33. Thus, even in this distance range the sample size needed to detect the syntenic marker on the usual analysis would have been about twice that required for equivalent power to detect a non-syntenic marker. A more detailed analysis of the general applicability of this method and its statistical power in relation to distance between modifier and major gene, will be presented elsewhere. Interestingly, myogenin (72.3 cM) which has been proposed as a downstream target of myostatin (LANGLEY et al. 2002; JOULIA et al. 2003) resides 4 Mb distal to the D1Mit262 microsatellite marker. Further studies are needed to determine whether myogenin may be the chromosome 1 modifier of Mstn. We thank Sóvári Krisztina, and Galli Györgyné for their excellent technical assistance. This research was supported by grant no. T043409 from Hungarian Scientific Research Fund (OTKA).
8
Mapping a syntenic modifier LITERATURE CITED FALCONER, D. S., 1960 Introduction to Quantitative Genetics. The Ronald Press, N.Y. GROBET, L., L. J. MARTIN, D. PONCELET, D. PIROTTIN, B. BROUWERS, et al. 1997 A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle. Nat. Genet. 17: 71-74. HALDANE, J. B. S., 1919 The combination of linkage values and the calculation of distances between the loci of linked factors. J. Genetics 8:299. JOULIA, D., H. BERNARDI, V. GARANDEL, F. RABENOELINA, B. VERNUS, et al. 2003 Mechanisms involved in the inhibition of myoblast proliferation and differentiation by myostatin. Exp. Cell Res. 286: 263-275. KAMBADUR, R., M. SHARMA, T. P. SMITH and J. J. BASS, 1997 Mutations in myostatin (GDF8) in Double-Muscled Belgian Blue and Piedmontese Cattle. Genome Res. 7: 910-916. LANGLEY, B., M. THOMAS, A. BISHOP, M. SHARMA, S. GILMOUR, et al. 2002 Myostatin inhibits myoblast differentiation by down regulating MyoD expression. J. Biol. Chem. 277: 4983149840. MCPHERRON, A. C. and S.-J. LEE, 1997 Double muscling in cattle due to mutations in the myostatin gene. Proc. Natl. Acad. Sci. USA 94: 12457-12461. SCHUELKE, M., K. R. WAGNER, L. E. STOLZ, C. HUBNER, T. RIEBEL, et al. 2004 Myostatin mutation associated with gross muscle hypertrophy in a child. N. Engl. J. Med 350: 2682-2688. SZABÓ, Gy., G. DALLMANN, G. MÜLLER, L. PATTHY, M. SOLLER, et al. 1998 A deletion in the myostatin gene causes the compact (Cmpt) hypermuscular mutation in mice. Mamm. Genome 9: 671-672. VARGA, L., Gy. SZABÓ, A. DARVASI, G. MÜLLER, M. SASS, et al. 1997 Inheritance and mapping of compact (Cmpt), a new mutation causing hypermuscularity in mice. Genetics 147: 755-764. VARGA, L., G. MÜLLER, Gy. SZABÓ, O. PINKE, E. KOROM, et al. 2003 Mapping modifiers affecting 9
Mapping a syntenic modifier muscularity of the myostatin mutant (MstnCmpt-dl1Abc) compact mouse. Genetics 165: 257-267.
10
Mapping a syntenic modifier Table 1. Summary of notation Mstn locus
C
Mutant deletion allele at Mstn
CK = MstnCmpt-dl1Ab
Wild type allele at Mstn
CB = +
ith Marker locus
Si
Marker allele derived from the Compact line
SiK
Marker allele derived from the CAST/Ei line
SiB
Modifier locus syntenic with Mstn
D
Modifier allele with positive effect on expressivity of the Compact trait
DK
Modifier allele with neutral effect on expressivity of the Compact trait
DB
Proportion of recombination between D and Mstn
rD
Proportion of recombination between marker Si and Mstn
rSi
Proportion of recombination between D and Si
rDSi
Frequency of allele DK in CKCK F2 population
pK
Frequency of allele DB in CKCK F2 population
qB
Frequency of allele SiK in CKCK F2 population
pi = (1-rSi),
Frequency of allele SiB in CKCK F2 population
qi = rSi
11
Mapping a syntenic modifier Standardized intensity of selection on DK
ID
Standardized intensity of selection on SiK, due to selection on DK
ISi
Change in frequency of DK due to selection for compact score
f(D)
Change in frequency of SiK, due to selection for DK
f(Si)
Male, Female with low compact score
M1, F1
Male, Female with high compact score
M5, F4
12
Mapping a syntenic modifier Table 2. The proportion of marker genotypes for the GPG groups, according to markers. Marker
S52 a
S304
S10
S309 S262 S133
S37
S154
GPG group genotype 0.120 b 0.369 0.566 0.631 0.670 0.816 1.010
1.120
SiBSiB
0.600 0.793 0.607 0.519 0.451 0.356 0.288
0.247
SiKSiB
0.363 0.200 0.342 0.393 0.447 0.488 0.498
0.498
SiKSiK
0.037 0.007 0.051 0.088 0.102 0.156 0.214
0.254
F1CBCB
SiBSiB
0.658 0.817 0.604 0.492 0.435 0.324 0.268
0.275
(313)
SiKSiB
0.304 0.173 0.348 0.435 0.466 0.545 0.537
0.489
SiKSiK
0.038 0.010 0.048 0.073 0.099 0.131 0.195
0.236
M1CKCK
SiKSiK
0.714 0.943 0.571 0.514 0.429 0.343 0.257
0.114
(35)
SiKSiB
0.200 0.057 0.343 0.343 0.429 0.400 0.571
0.629
SiBSiB
0.086 0.000 0.086 0.143 0.143 0.257 0.171
0.257
M5CKCK
SiKSiK
0.640 0.920 0.740 0.720 0.700 0.560 0.500
0.300
(50)
SiKSiB
0.360 0.060 0.220 0.260 0.280 0.400 0.400
0.460
SiBSiB
0.000 0.020 0.040 0.020 0.020 0.040 0.100
0.240
F1CKCK
SiKSiK
0.806 0.790 0.629 0.613 0.565 0.435 0.355
0.267
(62)
SiKSiB
0.177 0.210 0.306 0.306 0.371 0.468 0.468
0.500
M1CBCB (295) c
13
Mapping a syntenic modifier SiBSiB
0.016 0.000 0.065 0.081 0.065 0.097 0.177
0.233
F4CKCK
SiKSiK
0.595 0.881 0.548 0.500 0.500 0.476 0.286
0.238
(42)
SiKSiB
0.357 0.119 0.429 0.429 0.405 0.405 0.548
0.476
SiBSiB
0.048 0.000 0.024 0.071 0.095 0.119 0.167
0.286
M1CKCK
SiKSiK
0.714 0.943 0.571 0.514 0.429 0.343 0.257
0.114
(35)
SiKSiB 0.286 0.057 0.429 0.486 0.571 0.657 0.743
0.886
0.640 0.920 0.740 0.720 0.700 0.560 0.500
0.300
0.360 0.080 0.260 0.280 0.300 0.440 0.500
0.700
0.630 0.806 0.605 0.505 0.442 0.339 0.278
0.262
0.370 0.194 0.395 0.495 0.558 0.661 0.722
0.738
0.721 0.827 0.596 0.567 0.538 0.452 0.327
0.255
0.279 0.173 0.404 0.433 0.462 0.548 0.673
0.745
+SiBSiB
M5CKCK
SiKSiK
(50)
SiKSiB B
+Si Si
B
(M1+
SiBSiB
F1)CBCB
SiKSiB
(608)
+SiKSiK
SiKSiK K
(F1+F4)C C
K
SiKSiB (104) B
+Si Si
B
At six weeks of age the F2 mice were classified for muscularity by visual inspection using a five-grade scale from 1 (normal) to 5 (most muscular). The classification was always by G. M. Each animal was scored three times on the same day. An animal was characterized by the average of the 14
Mapping a syntenic modifier three independent scores giving 13 phenotypic categories ranging from 1.00, 1.33, 1.67, ..., to 5.00. Further details of the scoring procedure are in VARGA et al. (2003). DNA from tail tips was prepared using standard protocols. Eight microsatellites spanning chromosome 1 and distinguishing Comp9 and CAST/Ei were typed, using primers purchased from Research Genetics (Huntswille, AL). Nine loci (eight DMit microsatellites and Mstn ) covered chromosome 1 with an average genetic spacing of 11.1 cM (MGD). The cM positions of microsatellites were obtained from the MGD (Mouse Genome Informatics Project, The Jackson Laboratory, Bar Harbor, Maine, URL: http://www.informatics.jax.org, June, 2004). The largest intermarker distance was less than 19.7 (Table 2.). PCR, allele separation and silver staining were as described (VARGA et al.1997). a
Marker designation: D1Mit52 = S52, etc. Mstn is located at 0.277 M.
b
genetic map position of the marker in Morgans (MGD).
c
in parenthesis under GPG group, the number of individuals in the group.
15
Mapping a syntenic modifier Table 3. Chi-square comparisons of marker genotype frequencies by GPG groups. Total Comparison
S52 a
S304
S10
S309
S262
S33
S37
S154
M5CKCK vs
χ2 26.397
0.514
0.164
2.648 3.760
6.262
3.897
5.060
4.091
M1CKCK
***
M5CKCK vs
48.590 0.331
4.030
3.608 8.274 11.778 9.430 10.499 0.640
(M1+F1)CBCB
***
M5CKCK vs
19.662 1.049
2.389
3.043 3.331
3.645
1.579
4.280
0.346
(F1+F4)CKCK
**
M1CKCK vs
9.378 0.006
2.857
0.066 0.298
1.266
1.275
0.597
3.013
(F1+F4)CKCK
NS
M1CKCK vs
9.172 1.015
4.102
0.158 0.012
0.026
(M1+F1)CBCB a
0.002
0.072
3.791 NS
Marker designation: D1Mit52 = S52.
***P ≤ 0.001; **P ≤ 0.01
16
Mapping a syntenic modifier Table 4. Expected proportion of recombination between markers and Mstn according to map distance, and observed proportion of recombination between marker and Mstn locus and inferred intensity of selection at the marker, according to GPG group. S52 a
S304
S10
S309
S262
S33
S37
S154
Distance from Mstn (M) b
0.157
0.092
0.289
0.354
0.393
0.539
0.733
0.843
Expected Proportion of
0.134
0.084
0.219
0.253
0.272
0.329
0.384
0.407
recombination c Group
Observed proportion of recombination with Mstn
(M1+F1)CBCB
0.204
0.101
0.220
0.288
0.329
0.401
0.463
0.492
(F1+F4)CKCK
0.154
0.087
0.226
0.255
0.269
0.327
0.423
0.490
M1CKCK
0.186
0.029
0.257
0.315
0.357
0.457
0.457
0.571
M5CKCK
0.180
0.050
0.150
0.150
0.160
0.240
0.300
0.470
Intensity of selection M1CKCK:(M1+F1)CBCB
-0.111
-0.793
0.216
0.132
0.127
0.233
-0.024
0.316
M1CKCK:(F1+F4)CKCK
0.246
-0.730
0.177
0.316
0.448
0.591
0.139
0.324
M5CKCK:(M1+F1)CBCB
-0.148
-0.562
-0.408 -0.673 -0.766
-0.670 -0.656 -0.088
M5CKCK:(F1+F4)CKCK
0.200
-0.466
-0.434 -0.553 -0.554
-0.395 -0.504 -0.080
a
Marker designation: D1Mit52 = S52.
b
Calculated from the distances given in Table 2 by subtracting the location of Mstn (0.277 M).
c
Calculated using the standard HALDANE (1919) mapping function to convert map distance in
Morgans to proportion of recombination.
17
