GENOMICS
6,3%!-412
(1990)
The CEPH’ Consortium Primary Linkage Map of Human Chromosome 10 RAYMOND L. WHITE,* JEAN-MARC LALOUEL,* YUSUKE NAKAMURA,* HELEN DONIS-KELLER,t’* PHILIP GREEN,?* DONALD W. BOWDEN,* CHRISTOPHER G. P. MATHEW,~ DOUGLAS F. EASTON,~ ELIZABETH B. ROBSON,~~ NEWTON E. MORTON,B JAMES F: GUSELLA,# JONATHAN L. HAINES,# ANDRIES E. RETIEF,** KENNETH K. KIDD, t t JEFFREY C. MURRAY,*+ G. MARK LATHROP,*** AND HOWARD M. CANN***,* *Howard Hughes Medical Institute and Department of Human Genetics, University of Utah Medical Center, Salt Lake City, Utah 84 732; t Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63 110; *Collaborative Research, Inc., Bedford, Massachusetts 01730; glnstitute of Cancer Research, Sutton, Surrey SM2 SNG, England; “The Galton Laboratory, University College London, London NW1 2HE, England; YDepartment of Community Medicine, University of Southampton, Southampton SO9 4x/, England; #Neurogenetics Laboratory, Massachusetts General Hospital, Boston, Massachusetts 02114; **Department of Human Genetics, Faculty of Medicine, University of Stellenbosch, Tygerberg, 7505 South Africa; t t Department of Human Genetics, Yale University School of Medicine, New Haven, Connecticut 065 1o-8005; **Department of Pediatrics, Division of Medical Genetics, College of Medicine, University of Iowa, Iowa City, Iowa 52242; and ***Centre d’ftude du Polymorphisme Humain (CEPH), 75010 Paris, France Received
July 6, 1989;
revised
6, 1989
laborative effort, that of the CEPH collaboration to map the human genome (Dausset et al., 1990). The map was constructed from genotypic data generated from DNA of the 40 large nuclear families/pedigrees in the CEPH reference panel with probe and restriction enzyme combinations used by various laboratories participating in the collaboration. One laboratory provided data for the map from DNA of the CEPH reference panel plus 19 additional families. We also provide details of how the consortium map was prepared and analyzed. Human chromosome 10 has been described, cytogenetically, as a medium-sized, submetacentric chromosome, accounting for approximately 4.6% of the measured length of all the autosomes (Paris Conference, 1971). The mean genetic length of this chromosome in males, taken from an analysis of chiasma counts (Morton et al., 1982), is 127 CM. Linkage maps for chromosome 10, based on genotypes generated from the CEPH panel DNAs (or from other families) with probe and enzyme combinations from single laboratories, have already been published (Donis-Keller et al., 1987; Farrer et al., 1988; Lathrop et al., 1988, Nakamura et al., 1988a; Bowden et al., 1989). Recently the locus for a gene determining the disorder multiple endocrine neoplasia 2A (MENPA) has been assigned to chromosome 10 by linkage to already localized DNA polymorphisms (Mathew et al., 1987; Simpson et al., 1987). Genetic markers that have been used to improve
The first CEPH consortium map, that of chromosome 10, is presented. This primary linkage map contains 26 continuously linked loci defined by genotypes generated from CEPH family DNAs with 3’7 probe and enzyme combinations. C-genetic localization of some of the genetic markers indicates that the consortium map extends, at least, from lop13 to lOq26. The order of loci on the consortium map agrees with the physical localization data. The female map spans 309 CM (206 CM if an approximation of interference is included in the mapping function used to construct the map), and the mean genetic distance of intervals is 11 CM (7 CM). Also presented are maps of chromosome 10 from each of five CEPH collaborating laboratories, based on genotypes for all relevant markers in the CEPH database. The CEPH consortium map of chromosome 10 should be useful for localization of any gene of interest falling within the span covered. The genotypes in the chromosome 10 consortium map database are now available to the scientific community. o 1990 Academic Press,
November
Inc.
INTRODUCTION
We present here the CEPH consortium linkage map of human chromosome 10. This map represents a col’ CEPH, Centre d’Etude du Polymorphisme Humain. 2 To whom correspondence should be addressed at Centre d’Etude du Polymorphisme Humain (CEPH), 27 rue Juliette Dodu, 75010 Paris, France. 393
All
..
Copyright 0 1990 . _ . rlghta of reproduct!on
OSSEL7543/90 $3.00 by Academic Press, Inc. m any form reserved.
394
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the precision of localization of the MEN2A gene (Nakamura et al., 1989), enhancing prospects for diagnosis and isolation, are included in the CEPH consortium map. The localization on the consortium map of other markers closely linked to this gene will further refine its location. The CEPH consortium map also provides opportunities for localizing other genesof interest that reside on chromosome 10. MATERIALS
AND
METHODS
Genotypic Data Genotypes for 57 DNA polymorphisms, localized or possibly localized to chromosome 10, were generated from the CEPH,reference panel DNAs with probe and enzyme combinations used in nine CEPH collaborating laboratories. These 57 DNA polymorphisms represent 43 loci (Table 1). Genotypes for a subset of 27 of these 57 polymorphisms were generated in the CEPH panel plus 19 additional families by one of the collaborating laboratories (Nakamura et aZ., 1988a). Genotypic data were sent in standard format on 5.25-in. IBM-PC compatible floppy disks to CEPH, where they were incorporated into a chromosome 10 database suitable for preparing appropriate parameter and pedigree files for linkage analysis and mapping. All of the genotypic data have been examined for parental exclusions. In most of the participating laboratories, genotypes are checked by comparing them with the original autoradiograms, routinely, for multiple recombinants over relatively small genetic distances and/or for marker pairs showing less than 15% recombination. Table 1 summarizes the 57 probe and enzyme combinations for which there are genotypic data in the chromosome 10 database. Eleven of these DNA polymorphisms possessfour or more alleles. Two or more probe and enzyme combinations detected RFLPs at each of five loci, RBP3, DlOSll, DlOS16, DlOS34, and DlOS43. It should be noted that probe cTBIRBP9 (system 23 in Table l), isolated from a human genomic cosmid library with a cDNA probe for RBP3 (Nakamura et al., 1988a), generated genotypes that showed recombination with those of three other RBP3 markers, CRI-CS762/HincII (recombinant individuals 134509, 135007, and 140806), CRI-CTlS/MspI (recombinant individual 135007), and CRI-CS768/TaqI (recombinant individuals 134507 and 134509). These genotypes have been confirmed on the original autoradiograms, and there is no evidence for misidentification or contamination of the DNA from these individuals at CEPH. Although some of the DNA polymorphisms listed in Table 1 have been localized to chromosome 10 by physical mapping, techniques, others have been assigned to this chromosome by linkage to one or more previously assigned markers. Note that 5 polymor-
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phisms (representing four loci) listed in Table 1 are not localized to chromosome 10 (systems 32, 35, 55, 56, and 57). When the chromosome 10 database was prepared, these 5 markers were considered to be assigned or possibly assigned to this chromosome. Subsequently, analyses performed by laboratories participating in the construction of the consortium map failed to demonstrate linkage of only these 5 markers with the other 52 markers in the chromosome 10 database. Consistent with this finding, assignment of these 5 markers to other chromosomes has been reported. Thus, of the 57 DNA polymorphisms in the chromosome 10 database, 52, representing 39 loci, are assigned to this chromosome. For a number of probe and enzyme combinations, the observed heterozygote frequencies presented in Table 1 are based on all parents tested in the laboratories rather than the parental genotypes found in the database. Some laboratories contributed genotypic data to the database for only informative families (i.e., at least one heterozygous parent), although parents of uninformative families had been tested. The last column in Table 1 indicates how many individuals of the CEPH reference family panel were genotyped for each probe and enzyme combination. The CEPH panel consists of 517 individuals who account for 530 positions in the 40 reference families, because some individuals appear in more than 1 family. Those probe and enzyme combinations typed in more than 530 individuals were used in one of the collaborating laboratories with the CEPH panel and 19 additional families. Eight probe and enzyme combinations were each tested in lessthan 200 individuals, and six of these detected polymorphisms in the complex locus RBP3 or DlOSll. Two probe and enzyme combinations, H4/MspI and H4/BgZII, were used to genotype CEPH families by more than one laboratory. Two hundred fifty-one individuals were typed in duplicate for H4/MspI by different laboratories, and two individuals showed discrepant genotypes. Discrepant results were found for a single individual in a set of 109 CEPH panel members who were typed twice for H4/BgZII by different laboratories. The frequency of discrepant genotypes approximates 1% for these systems. These discrepant genotypes were not included in the chromosome 10 database. Correction of Genotypes Each contributor of genotypes was sent a chromosome 10 database. After distribution, five genotypes were corrected in the database as follows: The genotypes for CRI-L647 (DlOSll) of CEPH panel individual 134411 and for CRI-CS931 (DlOSll) of individuals 134405 and 134411 were not used for construction of maps. The genotype for CRI-JlOl/HindIII (DlOS43)
CEPH
CONSORTIUM
LINKAGE
TABLE Summary
of CEPH
MAP
OF
CHROMOSOME
395
10
1
Chromosome
10 Consortium
Markers
System”
Probe
Enzyme
Markep
Localization
Number of alleles”
Heter.d
Number typed
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57
ILPR pTHHlOB-1 pYNZ156 pTHH54 pl-101 OS-2 pTBlO-171 pMCK2 pMHZ15 H4 H4 pCMM17-1 pCMM17-4 pTBlO-163 cTBQ4 cTBQ7 cTBQ-12 cTBQ16 cTBQ20 pMCT122-2 pEFD75 pEFD70-2 cTBIRBP9 cTB14-15 cTB14-16 lcTB1434 2cTB14-34 cTB1436 p9-12a zap32 CDCP EMBL3-93 hp4f 1 cmhl0-2 pL28-5 HOATl-5 H38 CRI-L368 CRI-L647 CRI-JM14 CRI-JlOl CRI-JlOl CRI-CS762 CRI-J125 CRI-J127 CRI-CS931 CRI-CS932 CRICS933 CRI-CT19 CRI-CS933 CRICS762 CRI-CS768 CRI-J170 VTR4 H31 M7 M7
L?gZI B&II MspI MspI %I Hind111 PVUII RsaI MspI B&II MspI MspI PVUII MspI MspI TaqI Td MspI MspI TuqI TuqI PVUII TuqI TaqI RsaI TaqI ToqI MspI %I Hind111 TaqI MspI BclI B&II XmnI MspI MspI Hind111 TaqI Hind111 HindIII EcoRI HincII HindIII H&III TaqI Hind111 BamHI MspI TaqI TW TaqI PstI EcoRI HindIII BamHI MspI
ILPR DlOS13 DlOS18 DlOS14 DlOS4 DlOS20 DlOS19 DlOS15 DlOS17 RBP3 RBP3 DlOS16 DlOS16 D10S22 DlOS27 DlOS28 DlOS29 DlOS30 DlOS31 DlOS36 DlOS25 DlOS26 RBP3 DlOS32 DlOS33 DlOS34 DlOS34 DlOS35 DlOS5 EGR2 CDCP D9S37 VIM DlOS39 D13S62 OAT DlOS37 DlOS12 DlOSll DlOS62 DlOS43 DlOS43 RBP3 DlOS44 DlOS45 DlOSll DlOSll DlOSll RBP3 DlOSll RBP3 RBP3 DlOS49 DlOS6 DllS348 D7S422 D7S422
lOp15-p14 10 10 10 lOq22-q23 10 lOq21.1-q22 10 lOpter-p13 10qll.2 lOq11.2 10 10 lOq21.1 10 lOpter-p13 10 10 10 10 10 10 lOq11.2 10 10 lOpter-ten lOpter-ten 10 lOq21.1 lOq21.1 lOq21.1 9 lop13 1Optercen 13 lOq26 lOq25.1 10 lOq11.2 10 10 10 lOq11.2 10 10 lOq11.2 lOq11.2 lOq11.2 lOq11.2 lOq11.2 lOq11.2 lOq11.2 10 lOq26 llq21-qter 7p11.2-q21.1 7p11.2-q21.1
2 2 2 3 3 3 2 7 2 2 2 2 2 3 2 8 2 2 2 2 6 4 2 3 5 2 2 2 2 2 2 2 2 2 2 2 2 9 5 3 2 2 2 2 2 2 2 5 9 6 2 2 3 9 2 2 2
0.09 0.38 0.26 0.62 0.54 0.61 0.48 0.30 0.52 0.20 0.32 0.41 0.31 0.58 0.50 0.96 0.48 0.46 0.45 0.27 0.60 0.52 0.41 0.64 0.80 0.49 0.46 0.55 0.39 0.10 0.42 0.35 0.41 0.32 0.32 0.35 0.45 0.66 0.50 0.63 0.21 0.38 0.30 0.45 0.31 0.44 0.43 0.26 0.26 0.33 0.33 0.21 0.59 0.63 0.37 0.45 0.36
456 523 480 556 594 572 725 732 516 532 721 689 699 612 722 488 640 557 663 719 603 631 604 524 383 581 575 510 343 162 326 342 300 186 422 355 321 293 214 430 210 224 282 325 301 180 373 92 137 135 25 87 348 333 302 371 357
’ Sequential database numbers of probe and enzyme combinations. * Official Human Gene Mapping Workshop symbols. ’ Number of alleles for the polymorphism detected by the probe and enzyme d Observed heteroxygote frequency (see text, Materials and Methods).
combination.
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of individual 1204 was changed from 12 to 22 and that for CDC2 of individual 142404 was corrected to 12 from 11. Linkage
Maps from Collaborating
Laboratories
Five laboratories prepared maps with genotypes from the chromosome 10 database using programs for multilocus linkage analysis, CRI-MAP (Donis-Keller et al., 1987), LINKAGE (Lathrop et al., 1985), or MAPMAKER (Lander et al., 1987), and/or for multiple pairwise linkage analysis, MAP (Morton and Andrews, 1989). Six maps were prepared by the five laboratories. These maps were circulated among all contributors of chromosome 10 genotypes (members of the consortium map committee),*and they were compared for locus order, statistical support of orders, genetic distances of intervals, and sex differences in recombination frequencies. The committee met to analyze and discuss these comparisons and plan the final consortium map. RESULTS
AND
DISCUSSION
The Consortium Map of Chromosome 10 Preliminary analysis showed that markers H31/ HindIII, M7/BamHI, M7/MspI, EMBL3-93/MspI, and pL28-5/XmnI (see Table 1) could not be assigned to chromosome 10 by linkage. Subsequently, H31 and M7 were reported to be assigned to chromosomes 11 and 7, respectively, by in situ hybridization (A. Retief, personal communication); they are now designated DllS348 and D7S422. The loci defined by EMBL3-93 and pL28-5 were reassigned to chromosomes 9 and 13, respectively, by analysis of human-rodent somatic cell hybrid cell lines; these loci are designated D9S37 and D13S62. With the exclusion of these five markers, there remain in the database genotypes for 52 probe and enzyme combinations, representing 39 loci, assigned to chromosome 10. These genotypes are the actual basis for the construction of the six chromosome 10 maps contributed by five collaborating laboratories. These maps are presented below (see Chromosome 10 Maps from Individual Laboratories). The chromosome 10 linkage maps from collaborating laboratories were quite useful for the preparation of the consortium map. The maps allowed us to assemble a subset of genetic markers likely to be placed uniquely on the consortium map. This subset consisted of 29 chromosome 10 loci (41 probe and enzyme combinations), most of which were localized on at least four individual maps with acceptable support. The genotypes for this subset of loci contained the corrections listed above for DlOSll and DlOS43 (see Materials and Methods); locus CDCP was not included in the subset. RBP3 was included in this subset; it was defined by a haplotype constructed from genotypes for seven
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probe and enzyme combinations including the original genotypes for cTBIRBPS/TaqI. Members of the consortium map committee were asked to determine the maximum likelihood order of those of the 29 loci that could be assigned unambiguously (odds of 1OOO:l or greater) on the map. All of the 29 loci could not be localized unambiguously on a map, and the order containing the most loci to be localized was accepted as a tentative chromosome 10 consortium map. This order contained 26 loci. The placement of DlOSll, one of the 26 loci on the tentative consortium map, is problematic. DlOSll could not be localized with a desired support value in five of the individual maps constructed, based on genotypes of all or many chromosome 10 loci in the database (seebelow, Chromosome 10 Maps from Individual Laboratories). For the localization of DlOSll on the tentative consortium map, the level of support depended critically upon the specific subset of loci used in the analysis. When genotypes for all of the remaining tentative consortium map loci were included, the odds for placing DlOSll decreased substantially. The decrease in support may reflect errors in the genotypic data for these additional markers, causing inconsistencies in the dataset being analyzed and leading to lower likelihood support values. As the odds favoring the given location on the consortium map for DlOSll appear to depend on which markers were used for the analysis, the localization is open to question. Accordingly, DlOSll is not included on the consortium map. Three additional loci (4 probe and enzyme combinations) were placed on the tentative consortium map, DlOS15, DlOS17, and DlOS43. Pairwise lod score and multilocus linkage analyses indicated that each of these loci did not recombine with a locus assigned to the map (RBP3, DlOS39, and DlOS12, respectively). Furthermore, each locus could not be localized with the usual support to any other position on the tentative consortium map. A distance of 2.7 CM for the interval between DlOS17 and DlOS39 is reported for the male map from one individual laboratory (Fig. 4). However, this map is only slightly more probable (by odds of 1.3:1) than one with no recombination between these 2 adjacent loci for both sexes (see discussion under Chromosome 10 Map from the Institute of Cancer Research, below). Furthermore, this map was constructed with all 39 chromosome 10 loci, while DlOS17 was added to the tentative consortium map containing a subset of loci from the database. Addition or exclusion of loci in multipoint map construction could account for the difference between the consortium map and that from the collaborating laboratory for the genetic distance of this interval. With the addition of DlOS15, DlOS17, and DlOS43 and the exclusion of DlOSll, the consortium map contains 28 loci (37 probe and enzyme combina-
CEPH
CONSORTIUM
LINKAGE
tions). The order of these 28 loci is shown in Fig. 1, the chromosome 10 consortium map. The consortium map order reflects near-unanimous committee agreement. For each of the 28 loci on this map, localization at the position indicated is agreed upon by at least four of the six maps from laboratories of representatives on the consortium map committee. Moreover, no locus omitted from the map is localized acceptably on more than three maps from the individual laboratories. With the component loci and their order on the consortium map established, sex-specific genetic distances of intervals were computed for the map by multilocus linkage analysis (no interference assumed) and multiple pairwise analysis (interference specified). The genotypic data used for these computations were corrected for a single genotype for marker DlOS43 (see Materials and Methods); other loci with corrected genotypes (DlOSll and CDCZ) were not included on the consortium map. Despite evidence for recombination with other RBP3 markers, cTBIRBP9 was considered a nonrecombining component of this complex locus for the distance computations. Both sets of sex-specific interval distances are presented in Table 2. The chromosome 10 consortium map contains 28 loci continuously linked from locus DlOS31 (inferred from the genetic data to reside on distal 1Op) to DlOS6 localized to lOq26 (Fig. 1). With multilocus linkage analysis, the mean interval distance for the female map, which spans 309 CM (Haldane), is 11 CM (Table 2). With multiple pairwise analysis using a mapping function that provides an estimate for interference, the female map spans 206 CM and the mean interval distance is 7 CM (Table 2). Table 2 shows that recombination frequencies are higher in females for at least 18 of the 27 intervals of the consortium map. Testing of the sex-factored pairwise lod scores for each interval on the map revealed a significant female excess in recombination frequencies for the interval DlOS49-DlOS34 (Xf = 6.80). Significant excesses of male recombination frequencies were found for intervals DlOS33-DlOS32 (X4 = 6.32), DlOS20-DlOS18 (x; = 7.76) (discussed in Nakamura et al., 1988a), and DlOS36-DlOS6 (XT = 7.55); a borderline excess was found for interval DlOS62-DlOS4 (x! = 4.02). Note that the first and third of these intervals with increased recombination in males are near and at the ends of the map, respectively, consistent with earlier observations for chromosomes 10, 12, 16, 17, and 19 (Nakamura et al., 1988a; O’Connell et al, 1987; Reeders et al., 1988; Nakamura et al., 1988b; Nakamura et al., 1988c). The sex difference for the most distal interval on 1Oq (DlOS36-DlOS6) is especially interesting, because it replicates the excess of male recombination frequency found in this region between DlOS36 and DlOS25 (Nakamura et al., 1988a).
MAP
OF
CHROMOSOME
10
397
Information concerning the physical localization of 17 loci in the chromosome 10 database, 12 of which are on the consortium map, is available (presented in Table 1, shown for selected loci in Fig. 1) (Bowden et cd., 1989; Smith and Simpson, 1989; Warnich et al., 1989). The order of the physical (cytological) locations agrees with the order of these loci on the consortium map. The comparison of physical and genetic orders here is not stringent because some of the physical locations overlap; e.g., that of DlOS34 could be anywhere on 1Op and that of DlOS19, localized to lOq21.1-q22.0, overlaps with those for DlOS22, DlOS5, and DlOS4. If one omits DlOS34 and DlOS19 from the comparison, physical and genetic orders still agree. As DlOS34 is localized to lop and RBP3 to lOq11.2, the centromere may be placed between these adjacent loci, in an interval of 8.7 CM or less. Loci RBP3 and DlOS5 have been shown to be linked to the locus of the gene determining MENBA (Mathew et al., 1987; Simpson et al., 1987). It has already been suggested that markers DlOS15, DlOS34, and DlOS30 (not included in the consortium map) will be useful for the localization of the MEN2A gene (Nakamura et al., 1988a). Indeed, recent evidence indicates that the MENZA gene lies between DlOS34 and RBP3/DlOS15, in the pericentromeric region (Nakamura et al., 1989). Consortium and individual maps confirm the significance of these markers and suggest that DlOSll should also be considered for improving the precision of localization of this disease-determining gene. This consortium map of 28 loci should be useful for primary localization of any gene of interest falling within the region of chromosome 10 covered. A subset of 15 marker loci, 14 from this map (DlOS31, DlOS33, DlOS35, DlOS28, VIM, DlOS49, RBPS, DlOS22, DlOS16, DlOS14, DlOS62, DlOS20, DlOS12, DlOS27, and DlOS6), chosen for informativeness (9 of the 15 loci possess three or more known alleles) and spacing along the map, should permit detection of linkage with most genes on chromosome 10 which determine inherited disorders, given sufficient meioses from appropriate families, e.g., those in which a disease-determining gene segregates. The basic strategy used to construct the consortium map involved ordering a subset of chromosome 10 loci likely to be uniquely localized with an acceptable level of support. Maps constructed with the chromosome 10 database by laboratory groups participating in the consortium map project enabled us to determine which loci were likely to be uniquely localized. The disadvantage of this strategy was the exclusion of 11 chromo‘Locus DlOS27 is not on the final consortium map, but it has been mapped on the preliminary maps. It is included in this subset of markers because it is located in the Bame region as and more informative than DlOS36.
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398
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FEMALE
IlOS35
-
mS39Jms17 llOS28
15
MALE
14
DIOS31 13
D10S33
‘IM
12.3
D10S32
12.2 12.1
DlOS35
)lOS49
I 11.2
:
DlOS14 DlOS62
)lOS34
)lOS22 D10S29
>I0519 >I0516
DlOs14 22.1 22.2 22.3
l-4 1
1
DlOS29
D10S62
DlOS4 D10S36
23.1 23.2
D1OS1891omO
23.3
DICE+6 I
-
24.1 24.2
25.1
&QpMOs43
25.2 25.3
DlOS44
26.1
DlOS36 DIOS6
26.2 26.3
10
CEPH
CONSORTIUM
LINKAGE
some 10 loci from the consortium map. The alternative approach would have been to place on a map all of the 39 chromosome 10 loci at their most likely positions. In this case the drawback would be some regions with uncertain locus order. Both strategies have their merits, and it is useful to consider the reason(s) for constructing a genetic map before embracing or rejecting a particular approach. If the reason for constructing a primary map is to use it to localize genes of interest, unambiguous localization of the component markers is of paramount importance. The main motivation for organizing the CEPH collaboration to genetically map the human genome was (and still is) to use the resultant maps of chromosomes to localize genes of interest, essentially those that determine inherited diseases. This consideration, thus, underlies the strategy used to construct the CEPH consortium map of chromosome 10. Chromosome
10 Maps from Individual
Laboratories
Six maps of chromosome 10 contributed by five CEPH collaborating laboratories participating in the construction of the consortium map are presented here. The contributing groups were from the Howard Hughes Medical Institute at the University of Utah (laboratory 1); the Department of Genetics, Washington University School of Medicine (laboratory 2); the Galton Laboratory (laboratory 3); the Institute of Cancer Research (laboratory 4); and the Neurogenetics Laboratory, Massachusetts General Hospital (laboratory 5). These maps provided useful information, especially on locus order, which helped guide the construction of the CEPH consortium map of chromosome 10. Each of the maps was constructed from the data in the chromosome 10 database (containing genotypes for 57 probe and enzyme combinations representing 43 loci), and laboratories 2,3,4, and 5 used the corrections for five genotypes, as indicated above (see Materials and Methods), for the entire map construction process or in its final stages. Each laboratory used the original genotypes for marker cTBIRBP9 except for laboratory 3, which elected to avoid recombination within the RBP3 locus, as described under the section contributed by that group. When the five laboratory groups found that 4 loci (5 probe and enzyme combinations), detected by probes EMBL3-93, pL28-5, H31, and M7 (Table l), could not be linked to any of the other 39 loci in the chromosome 10 database and/or had been reassigned to other chromosomes, they excluded them from further consideration. The task for each group, then, was to prepare a map of the remaining 39 loci.
MAP
OF
CHROMOSOME
10
399
One or both of the basic strategies for map construction were used by each of the five laboratories. Laboratories 1, 2, and 5 constructed maps of chromosome 10 containing only unambiguously localized loci as determined by an acceptable support criterion for localization (Figs. 2,3, and 5). Laboratories 2,3, and 4 constructed maps with all of the 39 chromosome 10 loci placed at their most likely positions and used measures of local support to test for order (Fig. 4 and Tables 3, 4, and 5). Each of the five laboratories constructed a map with one of three programs for multilocus linkage analysis. Laboratory 3 also used the MAP program for multiple pairwise analysis to construct a map, building a trial map from data in lod score tables for all possible pairs of chromosome 10 loci. This map was then refined until all loci were at their most likely positions (described below under Chromosome 10 Map from the Galton Laboratory). The multilocus linkage analysis programs sequentially incorporate loci into a starting map. In each case, loci were incorporated, one by one, into a starting map, on the basis of a previous publication (laboratory 1) or consisting of an initial pair (laboratories 2 and 4) or larger subset (laboratories 3 and 5) of loci. A likelihood criterion is used to distinguish possible placements of the locus being added to the map. In general, the laboratories used an odds ratio of 1OOO:l or greater to uniquely localize a marker on the map and to distinguish between two orders of loci. Laboratory 1 found very stringent criteria of support for loci on the initial map (see Fig. 2) and localized additional loci with a minimal odds ratio of 1OO:l. Additional (local) support for a given order was computed by some of the laboratories as the likelihood difference(s) obtained from permuting the positions of two or more adjacent loci (see Fig. 2 and Table 3). For the maps constructed by laboratory 3, measures of local support were used to indicate uncertainty of order with respect to adjacent pairs of loci (Tables 4 and 5). Linkage maps can be depicted in various ways, as is evident from Figs. 2, 3, 4, and 5 and Tables 4 and 5. The maps consisting of uniquely localized loci indicate possible positions of ambiguously placed loci (Figures 2,3,4, and 5 and Table 6). Maps presenting an optimal order for the entire set of (39) chromosome 10 loci show support for permitted local permutations within that order (Tables 3, 4, and 5) or distinguish uniquely localized loci from the others (Fig. 4). Three maps present interval distances as recombination frequencies (no crossover interference assumed; Figs. 2 and 5) or cen-
FIG. 1. CEPH consortium linkage map of human chromosome 10. Two maps are shown, one based on female recombination frequencies and the other on male recombination frequencies. Interval distances shown on these maps are proportional to centimorgans. Loci shown on the same line are not separable by recombination. Recombination frequencies and the distances (centimorgans) for intervals are presented in Table 2. Physical localizations of selected map loci to regions on chromosome 10 are shown.
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TABLE Recombination
Frequencies Multilocus
(0) and Centimorgans:
DlOS31 DlOS33 DlOS32 DlOS35 DlOS39/DlOS17’ DlOS28 VIM DlOS49 DlOS34 RBP3/DlOS15’ DlOS5 DlOS22 DlOS19 DlOS16 DlOS14 DlOS29 DlOS62 DlOS4 DlOS20 DlOS18 D10S12/D10S43’ OAT DlOS44 DlOS36 DlOS6 Total
Chromosome
10 Consortium
analysis
Multiple
e Locus
2
Female
Male
Female
0.09 0.03 0.16 0.09 0.08 0.18 0.20 0.15 0.08 0.08 0.11 0.08 0.14 0.12 0.08 0.11 0.07 0.14 0.00 0.30 0.04 0.06 0.17 0.02
0.08 0.12 0.14 0.06 0.05 0.09 0.05 0.05 0.02 0.05 0.00 0.08 0.08 0.11 0.02 0.03 0.12 0.09 0.16 0.06 0.07 0.00 0.13 0.22
9.9 3.1 19.3 9.9 8.7 22.3 25.5 17.8 8.7 8.7 12.4 8.7 16.4 13.7 8.7 12.4 7.5 16.4 0.0 45.8 4.2 6.4 20.8 2.0
8.7 13.7 16.4 6.4 5.3 9.9 5.3 5.3 2.0 5.3 0.0 8.7 8.7 12.4 2.0 3.1 13.7 9.9 19.3 6.4 7.5 0.0 15.1 29.0
309.3
214.1
n Centimorgans computed from recombination frequencies b See Refs. (10) and (11). ’ Loci separated by / were not separable by recombination.
Male
by the mapping
timorgans computed from recombination frequencies (no interference) by the Kosambi mapping function which partially corrects for the effects of interference (Fig. 4). The interval distances are cumulated, starting from the most terminal pter marker, for the maps in Tables 4 and 5, these distances being, respectively, centimorgans computed with the Kosambi function and those derived from recombination frequencies estimated with a mapping function which specifies interference. Accordingly, the intervals and total length for the map presented in Table 5 tend to be shorter than those for the other maps. Figure 3 presents a map of uniquely ordered loci, with possible placements for other loci, but does not specify interval distances. As there is evidence for variation in recombination frequencies by sex, the maps in Figs. 4,and 5 and Tables 4 and 5 present sex-specific genetic distances. Chromosome 10 Map from the Howard Hughes Medical Institute, University of Utah. The 57 genetic systems available were reduced to 45 loci after haplotypes
analysis
eb
CM”
distance
pairwise
Map
function
CM*
Female
Male
Female
0.07 0.02 0.12 0.06 0.06 0.14 0.13 0.12 0.06 0.07 0.08 0.07 0.07 0.15 0.05 0.08 0.07 0.10 0.03 0.20 0.01 0.08 0.14 0.03
0.06 0.11 0.08 0.05 0.05 0.03 0.05 0.03 0.00 0.03 0.02 0.05 0.06 0.05 0.02 0.01 0.11 0.06 0.08 0.08 0.06 0.00 0.11 0.18
6.8 2.5 12.8 6.2 6.0 14.6 13.6 11.7 5.7 7.2 8.1 6.9 7.4 15.3 5.5 7.9 6.8 10.6 2.9 21.4 1.1 8.4 14.1 2.7 206.1
Male 6.1 11.0 8.1 5.1 4.8 3.1 4.8 3.1 0.0 3.1 1.7 5.5 6.0 4.6 1.8 1.5 10.7 6.3 7.9 8.4 5.6 0.0 11.4 18.9 139.7
of Haldane.
were constructed, defining the complex systems DlOS16 (probe and enzyme combinations 12, 13), DlOS34 (26, 27), DlOS43 (41, 42), RBP3 (10, 11, 43, 49, 51, 52), and DlOSll (39, 46, 47, 48, 50) (see Table 1). System 23 (CTBIRBPS), which shows recombination with other markers that also define RBP3, was not included in the haplotype for this complex system. Further reduction was obtained by deleting five systems (32, 35, 55, 56, 57) for which we could not find significant linkage to other systems. To construct a continuous map of chromosome 10, we elected to take advantage of a previously established primary map (Nakamura et al., 1988a). Because these loci originated from our laboratory, the original autoradiograms were available for data checking; moreover, most of these markers were tested on an expanded reference panel, and they appeared to span most of the chromosome. This map included 21 loci. Although most loci were suitably spaced and ordered with well-supported likelihoods, a few clusters of closely linked
CEPH
CONSORTIUM
LINKAGE
MAP
OF
CHROMOSOME
401
10
Initial Map 23 075
57 075 I
89 143 I
16 105 I
164 274 I
41 121 I
105 ooooomoooooww
45 082
I
70 095
I
77 128
85 183
I
125 190 11111111:1111111111111111
I
18 108
I
16 114
I
42 271
I
16 154
I
17 180
I
055 104 11111111:1111111111111111
DlOSll
L647
,
, TBIRBP9
RBP3
, TBQ16
DlOS30
271 owooooooooooowooooooooooowoooo 230 33333333333333333333333:333333333 128 oooooO0oooooOW
I
183 000000000000000
174 081 4U444444:4au44uu4~
100
I
I
$5bb '+a , I CDCZ
J127
rap32
DlOS45
DlOS62
JM14
, L EFD70
EGR2
,
,
H36
DlOS26
DlOS37
FIG. 2. Chromosome 10 map from the Howard Hughes Medical Institute, University of Utah. Initial map: The top row of figures represents 2 In likelihood support of order shown on the map for adjacent pairs of loci versus the inverted order. The second row of figures presents the recombination frequencies (X10’) for the intervals on the map, estimated under the assumption of no sex differences for recombination frequencies. Final map: Odds for the position shown for each locus on the map are $009 or greater than those for any other position. Each locus that could not be placed on the map with this criterion of support is shown under the map at its most likely position. Reiterated digits indicate the iteration during which a previously unlocalized locus was positioned on the map (e.g., 2222222 indicates the second iteration). Recombination frequencies shown above reiterated digits are those estimated for those map intervals receiving previously unlocalized loci during the indicated iteration. The full DlOSll haplotype is indicated here by the index probe, (CRI-)L647.
markers presented ambiguities. For each such cluster, we selected the most informative marker. Thus 16 anchor loci were available and strong support for their reported order was confirmed. There remained 24 loci to place on the map. Because the addition to a map of loci for which only poor support can be achieved complicates map construction inordinately, further mapping was performed with the program LINKAGE (Lathrop et al., 1985) through the following iterative algorithm. At a given iteration, the loci consist of two sets, a set of mapped loci and a set of as yet unmapped loci. The mapped loci define elementary intervals with estimated recombination values and documented support. Addition of loci to this map proceeds through the following steps.
(1) Regional assignment: For each unmapped locus, we find the subset of elementary intervals where this locus might lie by computing location scores on the current map; the candidate region may have already been reduced at previous iterations. (2) Selection of candidate anchors: In each interval, we select the locus that has the greatest odds of belonging in that interval, with some condition on minimum support. (3) Validation: For each candidate anchor, we validate the order of flanking loci and retest likelihood for location in other intervals within certain odds by maximum likelihood estimation; when candidates fall in adjacent intervals, further validation is performed using candidate anchors jointly.
24
28
9
33
53
26
30
-
-
-
-
45
-
-
12
7-----------------------~-
30
7--------------------~-
73~-----------------~30
-
-
-
31
-
-
4
-
1245-----------------
-451230--------------
123045-----------------
-4530------------------
-3031------------------
31
30
-
-
_
7
-
_
14
-
-
-
-
-
2
-
-
40
-
-
5
-
~
6
-
-
3
--3637--------
2-------------
40
217--------------
17
-
-
37
-42
42
37
-
_
38
36-
37
36-----
-
-
42
Note. Locus orders that differ from the best order by permuting some successive triple of loci and that have relative odds (as indicated best order, of at least 1:lOOO. Numbering of loci is as in Table 1; haplotyped systems are represented by the index of one of the component
---------~---------------------------2122
--------------------_______________
-----------------------------
36
42
-
-
-
29
-
-
39
----------------------------
11-
8
-
*1*-----------------------__
-
11
26
8
--------------------________
18
11
-42-37-----
-
18
with Relative Odds of within lOOO-fold of Best Order: Chromosome from the Department of Genetics, Washington University
3
---------------~------------
-------------------____
--------------------
------------------
---------_-----____
------------_____
16
Orders
g-----------------------~~---~~~~
34
1---------~--------------------------
25
1----------~-----------------~~~---~~~
19
-------------___
-----34
19
1925
1
Locus
TABLE
-
-
-
36
-
_
-
15
21 22
-
-
--_
_
_
54
-
_
_
20
21
-
-
-
-
-
_
_
22
to the right of the row), probes (Table 1).
-
----
-
-
44
10 Map
with
54
54
-
-
-
-
~
-
21
respect
to the
1:50
1:79
1:4.4
1:l
1:l
I:1
1:l
1:l
1:740
1:54
1:40
1:66
1:340
1:2.7
1:l
1:830
1:17
1:830
1:620
1:27
P1.2
1:l
1:l
1:130
1:l
z P r
8
CEPH
CONSORTIUM
LINKAGE
(4) Map update: The new anchors are added and recombination frequencies are reestimated in intervals receiving new anchors. This process is controlled by progressively decreasing the minimum support required before inclusion of new anchors, from minimum odds of 10,000 to 1000 and 100. All loci that could not be assigned to the map with odds greater than 100 were deliberately not included, as only their likely location can be trusted, rather, we summarized support for their location along the final map. All mapping was performed without considering sex differences in recombination and under the simplifying assumption of no interference. Our mapping process is summarized in Fig. 2. It should be noted that since this map was generated, the following loci have been subjected to some revision: DlOSll (systems 39 and 46), and DlOS43/HindIII (41) and CDC2 (31), as indicated above (see Materials and Methods). This may account for some minor differences with mapping results reported by other individual laboratories. Chromosome 10 Map from the Department of Genetics, Washington University School of Medicine. We first constructed an initial map by sequential incorporation of loci, using the “build” option in CRI-MAP (Donis-Keller et al., 1987). Loci were sorted in decreasing order of their number of informative meioses, and the first pair of loci in this list (DlOS28 and DlOS33) was chosen as the nucleus of the map. The next remaining locus was then placed in each possible position with respect to the nuclear map, and the multilocus likelihood was computed for each resulting locus order. Any order having a lOOO-fold lower likelihood than some other order for the same loci was excluded. If the locus was uniquely placed by this criterion it was incorporated into the map; otherwise, the next locus in turn was considered. In either case, a list of the permissible (nonexcluded) orders was appended to an orders database. Each time a locus was added to the map the entire process was repeated, placing loci with respect to the new map. At each stage, to reduce the number of likelihoods to be computed, only those possible locus placements that were compatible with the results of the prior tests stored in the orders database were considered. This procedure resulted in an ordered framework of loci whose order is unique, in the sense that any other order of this set of loci is inconsistent with the orders database; in other words, any other order is inconsistent with the results of some likelihood test-usually based on a subset, or an overlapping set of loci, rather than on the framework loci themselves-at 1OOO:l odds. (Since these tests may include loci not in the framework, this does not imply that all alternative orders for the framework loci will necessarily have 1000-fold lower likelihoods than the framework order.) In addi-
MAP
OF
CHROMOSOME
403
10
tion, the information in the orders database indicates the possible placements for the remaining loci with respect to the framework. The corresponding likelihood for each of these placements was computed (using the “instant” option of CRI-MAP). A schematic diagram summarizing the results of this analysis is shown in Fig. 3. Since this type of representation does not indicate the possible placements of ambiguously placed loci in the same region relative to each other, and since joint analysis of the genotypic information for such loci can
Dl OS31 &R
Dl OS33
I:170
DlOS32
I:01
DlOS35 Eos17
Dl OS39
1:1
DlOS26 VIM Dl OS49 Dl OS34 RBP3 DlOSll Dl OS5 Dl OS22 DlOSl9 DlOSl6 D10S14 I:59
D10S13
Dl OS29
I:1
Dl OS62 DlOS4 Dl OS20 DlOSl6 DlOSl2 OAT
3
1 J :t I:10
D10S27
I:56
Dl OS44
-I
1
Dl OS36 DlOS6
1
3 i&253
+;26
FIG. 3. Chromosome 10 map from the Department of Genetics, Washington University School of Medicine. The map, shown on the left, contains uniquely placed loci localized with odds of at least 1OOO:l. Possible placements, and their relative odds, of other chromosome 10 loci with respect to the map (the “best” placement is that with 1:l odds) are shown on the right. Other placements for these loci are excluded by odds of at least 1OOO:l.
404
WHITE
ET
AL.
TABLE Chromosome
10 Map
from
the Galton
Laboratory:
4 CRI-MAP
Analysis
of CEPH Location
Chromosome (CM)
System
Probe
Enzyme
Locus
Female
Male
1 19 25 24
IL2R cTBQ20 cTB14-16 cTB14-15 cTBl436 pMHZ15 cmhlO-2 cTBQ7 hp4f 1 CRI-J170 cTBQ16 DlOS34 pMCK2 RBP3 DlOSll p9-12a pTBlO-163 pTBlO-171 CDCP zap32 DlOS16 CRI-J127 pTHH54 cTBQ-12 pTHHlOh1 CRIJMl4 pl-101 OS-2 pYNZ156 CRI-L368 H38 CRI-JlOl HOATl-5 CRI-J125 cTBQ4 pMCT122-2 VTR4 pEFD70-2 pEFD75
B&I MspI RsaI TaqI MspI MspI BglII TagI BclI PstI MspI Haplo RsaI Haplo Haplo TaqI Ms.1 PULL11 TaqI Hind111 Haplo Hind111 MspI TaSI B&II Hind111 TuqI Hind111 MspI Hind111 MspI Haplo MspI Hind111 MspI TaqI EcoRI PVUII TaqI
ILZR DlOS31 DlOS33 DlOS32 DlOS35 DlOS17 DlOS39 DlOS28 VIM DlOS49 DlOS30 DlOS34 DlOS15 RBP3 DlOSll DlOS5 DlOS22 DlOS19 CDCP EGR2 DlOS16 DlOS45 DlOS14 DlOS29 DlOS13 DlOS62 DlOS4 DlOS20 DlOS18 DlOS12 DlOS37 DlOS43 OAT DlOS44 DlOS27 DlOS36 DlOS6 DlOS26 DlOS25
0.0 0.0 6.5 9.2 23.9 32.0 32.2 41.0 60.2 82.2 106.2 112.7 119.2 119.2 124.4 131.3 142.1 148.5 155.6 155.6 162.6 167.1 180.5 187.8 192.1 202.8 210.1 227.0 227.0 258.8 258.8 258.8 261.7 270.6 282.7 303.4 306.8 306.8 306.8
0.0 79.9* 89.4 101.9 115.7 122.4 122.4 127.5 133.5 136.9 144.3 147.8 147.8 150.4 150.4 155.2 157.1 163.4 167.7 167.9 173.4 173.4 182.7 185.6 188.2 188.2 200.0 208.5 222.6 229.4 229.4 229.4 234.0 234.0 241.3 253.7 274.1 277.5 278.1
r”“g
341 16 33 53 18 [8
*1 * 29 14 I31
301 [ * 451 4 17 2 40 5 6 1338 37
*I 36 44 15 [fi 22
211
of intragenic haplotype construction. [ 1, intergenic * Note. *, see text for definition whose order may be in doubt. ?, areas of low local support. ’ Local support = log,,, likelihood difference when adjacent pairs are reversed. * See discussion in text concluding this section.
often more precisely localize each of them with respect to the framework, we also performed the following analysis, using the “flips” option of CRI-MAP. Loci were first placed in their most likely interval in the framework, to obtain a candidate locus order incorporating all of the loci. Then, within this order, all permutations of each consecutive triplet of loci were constructed and the associated likelihood was computed. Table 3 lists the locus orders having relative odds within lOOO-fold of the best order.
haplotype-a
10 Data
set of contiguous,
Local support”
0.59 6.52 12.97 16.21 9.77 5.22 15.67 4.94 8.13 0.64 4.07 0.10 9.95 13.06 15.08 1.82
?
?
?
?
17.85 23.45 2.77 1.93 3.05 10.38 11.59 4.54 13.88
? ?
4.45 4.46 7.12 9.63 6.22
closely
linked
loci
Subsequent analysis revealed an anomaly concerning the support for placing DlOSll relative to RBPS. Although the “build” run described above uniquely placed DlOSll in the position shown in Fig. 3 at 1000 to 1 odds, this level of support appears to depend on the specific subset of loci used to perform the likelihood test. In particular, when DlOSll is instead tried in each possible position with respect to the entire set of chromosome 10 loci (using the order shown in Table 41, the odds favoring this placement of DlOSll drop
CEPH
CONSORTIUM
LINKAGE
to only 74 to 1, the next most likely position being on the other side of RBP3 between DlOS34 and DlOS30 (all other locations are still excluded at more than 1000: 1 odds). This appears to explain why four of the five groups analyzing the data did not uniquely place DlOSll with respect to RBP3. This anomaly may be a reflection of data errors, as several multiple crossovers in this region are revealed by the “chrompic” option of CRI-MAP. We have chosen to adopt (in Fig. 3) the unique location for DlOSll resulting from the “build” run in the expectation that random errors are unlikely to produce odds of 1000 to 1. The dataset used for this analysis contained the five corrected genotypes listed under Materials and Methods. No attempt was made to remove apparent RBP3 intralocus crossovers involving data for the probe cTBIRBP9. All likelihoods were calculated assuming no crossover interference and sex-equal recombination fractions. No information derived from physical localization studies was used in the map construction process. Chromosome 10 Map from the Galton Laboratory. Two maps were constructed, one with the program CRI-MAP (Donis-Keller et al., 1987) and one with MAP (Morton and Andrews, 1989). The maps were constructed independently, but with some comparison at various stages. The goal in each case was to produce as complete a map as the data permitted. Both approaches used the same genotypic data, which included haplotypes constructed by hand for RBPS, DlOSll, DlOS16, DlOS34, and DlOS43; to avoid apparent intragenic crossovers, all RBP3 data from individuals 140806 and 135007 and cTBIRBPS/TaqI data from family 1345 were omitted. EMBL3-93 (system 32) had been excluded from chromosome 10 by physical mapping at this laboratory and was not considered further. Original (uncorrected) genotypes were used for most of the construction process for both maps, and corrected data (see Materials and Methods) were incorporated only in the final stages. In preparation for CRI-MAP analysis, MAPMAKER (Lander et al., 1987) was used to provide pairwise sex-specific lod score tables and to suggest possible linkage groups. From the latter, a uniquely ordered triplet (DlOSlS-DlOSlS-D10S14) was identified by CRI-MAP and used as the basis for subsequent maps. The linkage groups and the physical map from HGMlO (Smith and Simpson, 1989) provided likely candidates that were tested, one at a time, with CRIMAP’s “all” option for incorporation into the map. A log,, likelihood support of at least 3.0 was required for the addition of a locus to the map. When all loci had been tested, those not fitted were tried again. DlOS27 and DlOS29 could be fitted only at a minimum log,, likelihood of 2.0, and three loci (pL28-5/XmnI,
MAP
OF
CHROMOSOME
10
405
H31/HindII, and M7/BamHI-M7/MspI, subsequently shown to be assigned to other chromosomes) could not be fitted at all. At various points in the analysis, two or more loci were grouped together as an intergenic haplotype, using CRI-MAP’s haplotype file facility. The criteria used were that the log,, likelihoods for adjacent positions of the test locus differed by less than 0.5, the distance between the loci was zero in one sex and less than 5 CM in the other, and the log,, likelihoods for the proposed haplotype were greater by at least 3.0 than those for alternate positions of the test locus. Six such haplotypes were eventually established and incorporated into the map. CRI-MAP allows recombination within a haplotype defined in this way, and the map distances in Table 4 reflect this. The remaining loci (ILBR, EGRB, and DlOSll) were then placed into the map in their most likely positions and the order was tested by permuting triplets down the length of the map. This produced two more likely orders, and only two other orders within a log,, likelihood difference of 3.0 from the best map. This best order, now based on the data containing the corrected genotypes, was tested, and the resulting, marginally better order was taken as final. Sex-specific map distances were calculated for this order, and local support was measured by inverting adjacent pairs of loci (Table 4). For the preparation of the second map, pairwise lod scores, factored by sex, were obtained from CRI-MAP and used as input to the MAP program. After global support for each locus was calculated (Morton and Andrews, 1989), three (pL28-5/XmnI, H31/HindIII, and M7/BamHI-M7/MspI) were excluded from further consideration since they yielded values less than 0.5, while the support for the remainder ranged between 14 and 340. An initial trial order was obtained from MAP (using a seriation algorithm), corrected on the basis of known physical evidence, and then MAP’s bootstrap facility was used to improve this order, with interference specified by a mapping parameter of 0.35 (Morton et al., 1985). Successive improvements were made by the bootstrap algorithm which, by permuting adjacent doublets in the map and adopting those resulting changes that exceed a given log,,, likelihood difference, seeks to maximize the overall likelihood, terminating when no further improvements can be made. The log,, likelihood difference was set low (O.l), since the intention was only to maximize likelihood. During the refinement process, megaloci were defined by combining pairs of contiguous, closely linked loci whose order could not be established. This was necessary to prevent the bootstrap algorithm from entering an infinite loop, and as MAP does not recognize any recombination within a megalocus, the distance between the component loci is 0 CM (Table 5). Locus
406
WHITE
ET
AL.
whose positions can be uniquely established with support of log,, likelihoods of 3.0 or more. The high degree of similarity between the two maps is encouraging, since it shows that the resulting order is largely independent of the strategy used to obtain it. Local support, calculated by entirely different methods, is not directly comparable, but differences between the two maps occur in areas of low local support in at least one map. Where differences in order exist, in the region between DlOS49 and DlOS5 and in the region between DlOS18
order of the map shown in Table 5 was constructed using the uncorrected data; using the corrected genotypes did not change the order. Only the distances and local support (derived from map distance and standard error; Morton and Andrews, 1989) were recalculated with data containing the corrected genotypes. The two maps represent complementary strategies: building by stepwise addition and attempting to refine a trial map toward the maximum likelihood order. Neither of the two maps presented here contains only loci
TABLE Chromosome
10 Map
from
the Galton
Laboratory:
5
MAP
(13) Analysis@
of CEPH Location
System 11
Probe
Enzyme
Locus
Female
Chromosome
10 Data
(CM) Local supportc
Male
ILZR cTBQ20 cTB14-16 cTBl4-15 pMHZ15 cTB14-36
BglI MspI RsaI TaqI MspI MspI
IL2R DlOS31 DlOS33 DlOS32 DlOS17 DlOS35
0.0 0.0 5.3 1.8 25.1 18.5
0.0 O.Ob 7.2 19.2 31.9 27.2
2.9 6.3 7.1 3.2
cmhlO-2 cTBQ7 hplf 1 CRI-J170 DlOS34 DlOSll pMCK2
B&II TaqI BclI PstI Haplo Haplo RsaI
DlOS39 DlOS28 VIM DlOS49 DlOS34 DlOS15 DlOSll
25.1 31.1 44.3 57.1 72.1 78.3 73.9
31.9 36.7 39.7 43.9 45.7 47.5 46.8
6.0 4.8 4.3 9.2 3.4 1.0
18 29 14
RBP3 cTBQ16 p9-12a pTBlO-163
Haplo MspI TaqI MspI
RBP3 DlOS30 DlOS5 DlOS22
78.3 83.1 87.1 94.1
47.5 48.5 51.9 52.4
3.3 3.0 2.3
r3: 301 * 45 4 17 2 40 5 6 3 [E
pTBlO-171 CDC2 zap32 DlOS16 CRI-J127 pTHH54 cTBQ-12 pTHHlOh1 CRI-JM14 pl-101 OS-2 pYNZ156 CRI-L368 HOATl-5
PUUII TQ41 Hind111 Haplo HindIII MspI WI BglII Hind111 Td Hind111 MspI Hind111 MspI
DlOS19 CDC2 EGR2 DlOS16 DlOS45 DlOS14 D10S29 DlOS13 DlOS62 DlOS4 DlOS20 DlOS18 DlOS12 OAT
105.5 100.9 105.5 109.3 116.5 125.1 129.8 132.9 139.0 146.1 155.7 158.0 181.5 180.1
59.7 56.8 59.7 64.3 64.3 68.5 70.2 72.4 72.4 82.3 87.8 93.4 106.5 102.7
3.7 1.7 5.3 2.0 0.9 1.0 4.8 4.6 0.5 7.9 2.1
37
H38 CRI-JlOl CRI-J125 cTBQ4 pMCT122-2
MspI Haplo Hind111 MspI TaqI
DlOS37 DlOS43 DlOS44 DlOS27 DlOS36
181.5 181.5 187.9 194.0 209.4
106.5 106.5 106.5 111.1 120.2
1.8 3.5 8.7
?
VTR4 pEFD75 pEFD70-2
EcoRI TaqI PVUII
DlOS6 DlOS25 DlOS26
211.6 211.6
137.9 142.5 142.5
5.7 1.3
?
191 25 24 r289
341 16 33 53 * L*s
*I
*1 44 15 20 I5241
221
of intragenic haplotype construction. [ ] megalocus, Note. *, see text for definition be in doubt. ?, areas of low local support. o Interference is allowed for using a mapping parameter p = 0.35. b See discussion in text concluding this section. ’ Local support based on map distance and standard error, (w/SE,)*/4.605 (11).
a set of contiguous,
closely
linked
?
2.1 5.6
loci whose
order
?
? ?
?
may
CEPH
and DlOS44,
CONSORTIUM
LINKAGE
at least one of the maps has low local support in the area, suggesting that the two orders are indistinguishable at the given level of local support. With respect to the two different orders in the former region, note the low level of local support for the position of DlOSll with respect to RBP3 on the map shown in Table 4 and with respect to DlOS34 on the second map (Table 5) and the low local support for DlOS34 with respect to DlOS30, also on the second map. The order of OAT and the megalocus containing DlOSl2 is inverted between the two maps, and there is evidence for low and marginal local support in their region on the second map (Table 5). The differences arise from the strategies used and, rather than indicating conflict, can be regarded as indicating regions of uncertainty in the map. Regions of maximum uncertainty are highly likely to be at the ends of the map, and the results given here by the two methodologies illustrate this problem. The difficulty in localization at the pter end of the maps presented here is compounded by a poorly informative terminal marker, ILBR. Differences between these complete maps, containing regions of order uncertainty, and the smaller consortium map may, in general, be explained by the nature of the data, as in some cases inserting one of a pair of loci will exclude the other and vice versa. The interval distances calculated by MAP are smaller than those obtained with CRI-MAP (Tables 4 and 5). Accordingly, the overall, multiple pairwise, male map length of 143 CM is closer to the 127 CM estimated from chiasmata counts in spermatogenesis (Morton et al., 1982) than that obtained by the multipoint analysis. Chromosome 10 Map from the Institute of Cancer Research. We have constructed a linkage map of the chromosome 10 markers typed in the consortium map database, principally using the computer program CRIMAP. CRI-MAP provides rapid computation of multipoint likelihoods, particularly for CEPH-type nuclear families, enabling large multipoint maps to be constructed (Donis-Keller et al., 1987). The 57 probe/enzyme combinations for which data were available included 43 distinct loci; corrected genotypes (see Materials and Methods) were used in constructing the map. Where more than one marker was available for the same locus, these were treated as haplotypes so that the markers were forced by the program to be placed consecutively, although not necessarily with zero recombination. In only one situation, however, was intralocus recombination observed; the cTBIRBP9 probe was recombinant with other markers at the RBP3 locus and is shown separately above RBP3 on the map. We first used the “build” option in CRI-MAP to search for the largest set of loci that could be uniquely
MAP
OF
CHROMOSOME
10
407
ordered with a likelihood ratio of at least 1OOO:l over other orders, adding loci successively to an initial linked pair of loci (DlOS33 and DlOS28). By this process 25 loci could be uniquely ordered-these correspond precisely to the loci ordered on the tentative consortium map before addition of DlOS15, DlOS17, and DlOS43. Note that in arriving at this ordered set of loci, the likelihoods calculated utilized ordering information from other markers not uniquely ordered, but placed in their various possible positions. This use of additional markers to provide indirect evidence of ordering allowed several more markers to be uniquely ordered than would otherwise have been possible. Likelihoods for possible placements of the remaining loci within this definitive order were then computed. The possible positions of each locus, with likelihoods of not smaller than l/lOOOth of the best order, are shown in Table 6. At this stage, 4 loci (H31, M7, EMBL3-93, and pL28-5) were discovered not to be placeable anywhere within the map, nor were they linked to any of the loci near the ends of the map. These loci were therefore discarded from the analysis, leaving 14 to be placed on the map. By reducing the tolerance of placement to lOO:l, and by checking likelihoods of various possible positions of loci in particular regions, it was possible to arrive at a maximum likelihood map for all 39 loci. As a check on this map, likelihoods were calculated for all orders obtained from this order by permuting adjacent triples of loci to search for orders with higher likelihood. In addition, for loci such as DlOS30 and DlOS27 which could be placed in two intervals that were not contiguous, complete maps were calculated under different assumed positions to check that the position with highest likelihood had been identified. Likelihoods for these final positions of the loci not definitely placed, compared to their next most favored placement, are shown in Table 6, and the resulting map is shown in Fig. 4. A male distance of 2.7 CM was found between DlOS39 and DlOS17 on this map. This distance, however, is uncertain, since DlOS39 is poorly informative. In fact, when the order of these two loci is inverted, the resultant map shows estimated sex-specific recombination frequencies of 0 for the interval and is only slightly less probable than the map presented here (odds of 1:1.3). Accordingly, we do not disagree with the placement of DlOS17 at the same position as that of DlOS39 on the consortium map. The genetic distances between adjacent loci on the map assume that recombination fraction and genetic distance are related via the Kosambi function. The total length of the map is estimated to be 275.8 CM in males and 309.8 CM in females. However, it is likely that these distances are overestimates for the following reasons: First, the dataset is likely to contain an unknown
408
WHITE
TABLE Possible
Positions
Locus
of Loci
Most
ET
AL.
6
Not Uniquely Ordered at 1OOO:l Odds on Chromosome from the Institute of Cancer Research
likely
interval
ILPR
[pter,
DlOS17 DlOS30
[DlOS39, [DlOS49,
DlOS28] DlOS34]
1.3:1 6.8~1
DlOS15 DlOSll EGRP
[RBP3, DlOS5] [RBP3, DlOS5] [DlOS22, DlOS19]
1:l 26:l 1.3:1
CDC2 DlOS45 DlOS13 DlOS37
[DlOS19, [DlOS19, [DlOS29, [DlOSlS,
DlOS16] DlOS16] DlOS62] DlOS12]
59:l 17:l 41:l 1:l
DlOS27 DlOS43 DlOS26 DlOS25
[DlOSlS, [DlOS12, [DlOS6, [DlOS6,
DlOS12] OAT] qter] qter]
17:l 1:l 2.0:1 2.0:1
D Ratio of likelihood obtained * Positions of the locus giving
by placing likelihoods
DlOS31]
Support”
locus in most likely interval, not smaller than l/lOOOth
number of erroneous typings, resulting in a spuriously high number of recombinant events. Interval distances between neighboring loci on the map are typically only a few centimorgans, so that most meioseswill not produce a recombination between a given locus and its nearest neighbor. Typing errors at a locus are therefore more likely to introduce a spurious recombinant than to eliminate a real one. Indeed, by using the “chrompic” option in CRI-MAP, a few individuals with implausibly large numbers of recombinant events could be identified. Second, the male map is inflated by the very large estimate for the distance between ILZR and DlOS31. This distance is extremely uncertain due to the poor informativeness of ILBR, and given the female recombination frequency of 0 it is reasonable to supposethat the true distance will be much smaller. Third, the estimated genetic distances on this map are much greater than would be suggestedby data on observed chiasmata distributions. Thus the distances for this map should be used only as a rough guide. Chromosome 10 Map from the Neurogenetics Laboratory, Massachusetts General Hospital. The corrected genotypic data for the 57 probe and enzyme systems were extracted from the CEPH chromosome 10 database into files conforming to the LINKAGE format. These files were converted into LIPED format files compatible with the LIPIN (Trofatter et aZ., 1986) data management system. Further conversion was necessary to create files acceptable to MAPMAKER (Lander et al., 1987). Data were checked at each stage of the conversion to ensure that no errors were introduced.
3.8:1
Other
10 Map
possible’
intervals
[DlOS31, DlOS33] [DlOS33, DlOS32] [DlOS35, DlOS39] [DlOS34, RBPB] [RBPB, DlOS5] [DlOS34, RBPB] [DlOS34, RBP3] [DlOS19, DlOSlS] [DlOS16, DlOS14] [DlOS22, DlOS19] [DlOS16, DlOS14] [DlOS14, DlOS29] [DlOS12, OAT] [OAT, DlOS44] [DlOS44, DlOS36] [DlOSlS, DlOS12] [DlOS36, DlOS6] [DlOS36, DlOS6]
divided by likelihood for the next best position. of the likelihood for the best position.
MAPMAKER was chosen as the analysis tool for two reasons. First, it can be faster than LINKAGE for calculations involving a large number of loci. Second, no other group participating in the consortium map was using MAPMAKER, thus allowing a diversity of analytic tools to be used on the same dataset. Our general approach for construction of the map proceeded as follows: First, all possible two-point lod scores were calculated from the genotypes for the 57 systems in the chromosome 10 database. (It was at this point that the loci defined by EMBL3-93, pL28-5, H31, and M7, later found to reside on other chromosomes, were discarded from further analysis, because they did not have substantially positive lod scores with any other markers in the database.) Superficial examination of two-point lod scores generally will reveal three to five very informative loci (determined by correspondingly high lod scores) residing in the samegeneral chromosomal region. All possible orders of these loci are tested, and a unique order (defined as a log,, likelihood difference of 3, odds of 1OOO:lor better, favoring this order) is defined. If no unique order is found, new starting loci are chosen. Additional loci are then tested in all possible positions against this defined map and are added if they have one position favored by odds of at least 1OOO:l. The resulting chromosome 10 map is presented in Fig. 5. Examination of the two-point lod scores allowed us to choose five loci (VIM, DlOS28, DlOS35, DlOS32, and DlOS33) that appeared to be spaced approximately 10 CM apart. All possible orders of these five loci were
CEPH
CONSORTIUM
LINKAGE
then tested. A log,, likelihood difference of 3.8 was found favoring a single order (Fig. 5). Additional markers were added to the map sequentially by testing them in every possible position against the established map. In all, 25 loci could be uniquely placed on this map. These loci define 21 unique positions, as DlOS12, DlOS37, and DlOS43 map to a single location, DlOS44 and OAT to another single location, and DlOS15 and RBP3 to still another single location. However, many loci could not be placed distinctly within this framework. Usually, these loci could be placed in one of two or three positions (Fig. 5) and could be significantly excluded from a large portion of the map. Because cTBIRBP9 demonstrated recombination with other RFLPs at the RBP3 locus, it was considered separately in determining locus order of the map. However, due to the small number of recombination events, it could not be placed in a unique position relative to the RBP3 locus (Fig. 5). Several points are worthy of consideration. First, MAPMAKER constructed a map similar to that generated by CRI-MAP and the LINKAGE package. However, MAPMAKER, as currently released, does not encourage users to examine sibships where either one or both parents are missing genotypic data, which occurred multiple times in this dataset. In fact, in the final analyses we were able to place some loci only after entering inferred parental data by hand. Unfortunately not all of the missing parental genotypes could be inferred, and thus only a subset (albeit a very large subset) of data could be analyzed. Some differences between our map and those of other laboratory groups are interesting. Two loci (DlOS14 and DlOS29) were placed with greater than 1OOO:l odds in a position inverted relative to the other maps. We are not entirely sure why this occurred, but it may indicate that the subset of data that MAPMAKER uses favors this alternative order, and thus may indicate internal inconsistencies within the entire dataset. Most other maps were able to uniquely localize several additional consortium map loci, including DlOS31, DlOS39, DlOS17, and DlOS34. These loci could not be placed on our map with the requisite 1OOO:l odds; in all cases they mapped to the positions given in the other maps with odds of at least 1OO:l. Our reduced level of support most likely arises from MAPMAKER not fully considering the overall dataset. Similarly, we observed no recombination between OAT and DlOS44 on multipoint analysis, probably a result of the data reduction. Second, the speed with which MAPMAKER can generate these maps can be truly impressive. On a VAX 8700, the 1596 two-point lod scores were calculated in less than 1 min, and the results easily displayed. Threeto five-point analyses generally took less than 3 min per order to calculate. However, several three- to five-
MAP
OF
CHROMOSOME
409
10
point analyses did take in excess of 30 min per order, depending on which markers were included in the analysis. This probably resulted from evaluation of phase-unknown intercross matings (both parents heterozygous for the same alleles) for the relevant loci. In all, the map was generated using approximately 4 days of CPU time on a VAX 8700. Finally, one concern that arises is that the mapping strategy we have employed, although generating an accurate map, does not necessarily generate the most inclusive map. An alternative set of initially mapped markers may allow a greater (or lesser) number of loci to be placed within a framework. This would depend on ambiguities of order (probably arising from an inevitable set of genotypic errors) being resolved by the removal of the offending locus. We have tested for a more complete set of mapped loci by trying different sets of starting markers and changing the order with which loci are entered into the map. This testing quickly became time-consuming and did not produce a more complete map. Comments
on Individual
Maps
This section summarizes various differences between the individual maps and the consortium map, and among the individual maps themselves, for locus order. In emphasizing differences here, it should be recognized that there is reasonable agreement among the individual maps and with the consortium map. The map from laboratory 1 (Fig. 2) contains DlOS27 and ILSR, whereas the consortium map does not. DlOS62 and DlOS6 on the consortium map are replaced by DlOS13 and DlOS25, respectively, on the map from this laboratory. These differences are explained by noting that DlOS27, DlOS25, DlOS13, and IL2R were not included in the subset of loci used to determine locus order on the consortium map; the first two loci were included on the initial map from laboratory 1. DlOS13 and DlOS25 are poorly localized on at least four individual maps (Figs. 3, 4, and 5 and Table 3), and DlOS27 is poorly localized on three maps (Figs. 3 and 4; see above, Chromosome 10 Map from the Galton Laboratory). ILBR, a poorly informative marker, was added to the final map from laboratory 1 because there was no recombination with DlOS31, the most pter marker on the initial map. ILBR is localized to the pter region of all of the other individual maps but its precise position on four maps is in doubt. Aside from the presence of DlOSll, the order of uniquely placed loci on the map from laboratory 2 (Fig. 3) is identical to that of the consortium map. The difficulty in placing this locus with respect to RBP3 on the consortium map is reflected in the inability to localize DlOSll with acceptable support on five individual maps, which may depend on which subset of loci
410
WHITE Female pter 77.6 9.5 12.5 13.9 4.7 2.7 4.5
ILiR I DlOS31 I D10S33 I DlOS32 I D10S35 I DlOS39 I DlOS17 I DlOS28
5.7
0.0 6.5 2.7 14.8 8.1 0.0 9.1 18.8
Viii
3.8 7.1 3.1 0.9 0.0 0.0 2.7 4.3 1.3 0.0
6.8
I
DlOS49 I DlOS30 I DlOS34
I
IRBP9 I RBP3 I DlOS15 I DlOSll I DlOS5 I D10S22 I EGR2
21.1 23.5 6.4 5.4 2.2 0.0 5.3 7.6 11.4 6.5
I
0.0
I
7.8
D10S19 4.0
CDC2 4.4
6.4 D10S45
2.2 9.4 3.0
6.8
I
DlOSl6 I DlOSl4
10.4
I
7.6
I
3.4
I
9.5
D10S29 2.1
DlOSl3 0.5 11.0 0.6 11.0 11.2 3.5 0.0 0.0 5.1
D10S62 I DlOS4
I
D10S20 I DlOS18 I DlOS27
I
DlOS37 I DlOS12 I DlOS43
I
16.8 0.0 0.0 3.9
15.5
I
2.2
I
0.0
I
0.0
DlOS6 DlOS26 0.6
32.8
I
D10S36 3.2
1.7
is used for its localization. For further discussion on this issue, seethe sections on the consortium map and the map from laboratory 2. Probably the most important difference found between the map from laboratory 5 and the consortium map is that of the order of two uniquely placed loci, DlOS29 and DlOSl4 (Fig. 5). The order on the former map is inverted with respect to that on the consortium map (and also on each of the other individual maps). This difference may result from the use of a (large) subset of genotypes from the CEPH families in the analysis by laboratory 5. As discussed in the section describing the map from this laboratory group, the program MAPMAKER usually omits data for analysis from families with one or both parents not genotyped. Another difference is the ambiguous localization of six consortium map loci on the individual map from this laboratory. Furthermore, DlOS37, DlOS27, and CDC2, which are not included on the consortium map, are uniquely placed on this individual map; in each case, at least three other laboratories were unable to incorporate these loci in their maps with acceptable support. These differences, too, may be attributable to the subset of genotypes used to construct the map from laboratory 5. It is difficult to compare the maps from laboratory 3 with the consortium map because, as noted above, different strategies were used to construct them. All 39 chromosome 10 loci were placed on the maps from laboratory 3 at their most likely positions, which were tested by measures of local support. Nonetheless, it is evident that the order of the 28 loci on the consortium map is identical to their order on these individual maps except for an inversion of (the megalocus containing) DlOS12/DlOS43 and OAT on the map prepared by multiple pairwise analysis. The group from laboratory 3 suggeststhat the observed difference reflects the uncertainty of the order in this region, as indicated by lbw levels of local support. None of the other maps from individual laboratories, including that from laboratory 3 prepared by CRI-MAP, show an order for DlOS12 and OAT different from that of the consortium map. Other possible evidence of uncertainty of order in this region is the ambiguous localization of DlOS37 (Figs. 2, 3, and 4) and of DlOS27 (Figs. 3 and 4).
10.1 DlilS44
21.2
16.8
AL.
OAT
0.0 13.5
8.7
ET
DlOS25
I
qter
FIG. 4. Chromosome 10 map from the Institute of Cancer Kesearch. The loci on this map are placed at their most likely positions, i.e., at positions giving the highest likelihoods. Loci shown in bold type could be uniquely ordered relative to one another, with odds of 1OoO:l or greater over other orders (see text). The estimated male and female genetic distances (in centimorgans) between adjacent pairs of loci are shown to the left and right of the figure. These distances are computed from recombination estimates by using the Kosambi mapping function.
CEPH Male
Female
CONSORTIUM
LINKAGE
MAP
OF
CHROMOSOME
411
10
pter I -----
.124
DlOS33
,036
.147
.151
.074
.116
.087
.197
1-
DlOS32
j-
DlOS35 DlOS28
.002
.247
.016
.176
.040
.112
.016
.llO
I-
I-
VIM
I-
DlOS49
) DlOS31 I
I 1 IL2R
; DlOSl7 I
RBP3/DlOS15
1DlOS30
IDlOS34
;DlOSll
I
I
1 I cTBIRBP9
DlOS5 I DlOS22 .027
.145
.119
,092
CDC2
1 EGRP DlOS16
.045
I
DlOSl9
.120
DlOS13
1DlOS45 I
DlOS29 .017
.046
.032
.190
.109
.045
. 100
.164
DlOS14 DlOS62 DlOS4 DlOS13 DlOS20 .136
.184
.073
.109
.054
.077
.004
.080
.305
.176
DlOS27 DlOS18 DlOS12/DlOS37/DlOS43 DlOS44/OAT
I -----
qter
DlOS6
i~l0~36
1DlOS25
I
I
I~l0~26
FIG. 5. Chromosome 10 map from the Neurogenetics Laboratory, Massachusetts General Hospital. The map represents all loci that could be placed with odds of 1OoO:l or better against every other position in the then current map. Loci to the right of the map have at least two positions, represented by the vertical lines, whose odds are within 1OOO:l of each other. All other positions for these loci are excluded with odds greater than 1OOOzl. The estimated male and female recombination fractions between adjacent pairs of loci, computed under the assumption of no interference, are shown to the left of the map. Loci that map to the same location (because no recombination was observed by two-point or multipoint analyses) are separated by a /.
The maps from the five contributing laboratories show differences in the region flanked by DlOS49 and DlOS5. All six individual maps agree with the order DlOS49, RBP3/DlOS15, and DlOS5 in this region. Additionally, DlOS34 was localized in the sameposition in this region on four maps (Figs. 2,3 and 4 and Table 5), giving an order agreeing with that of the consortium map (see Fig. 1 and Table 2). DlOSll was localized in this region, between RBP3/DlOS15 and DlOS5 (Fig. 3)) with acceptable support by a single laboratory. The two maps constructed by laboratory 3 placed DlOS30
within this region. However, the maps differed in the most likely position for this locus (Tables 4 and 5). Taken together, the maps from all the collaborating laboratories identify this region as one of uncertain order, especially with respect to the placement of DlOSll and DlOS30. Availability of CEPH Chromosome 10 Probe and Enzyme Genotypes With the publication of this CEPH consortium map, the genotypes in the chromosome 10 database are now
412
WHITE
available to the general scientific community. database may be obtained by contacting CEPH.
The
ACKNOWLEDGMENTS Author lists somehow do not succeed in completely conveying who contributed. In the case of the laboratories that collaborated in preparing the CEPH consortium map of chromosome 10, the difficulty in giving credit where it is due is compounded. Data collection and analyses would not have been possible without the scientific involvement and work of the following colleagues: at the Galton Laboratory and Imperial Cancer Research Fund Laboratory-Vince Andrews, John Attwood, Sue Povey, and Nigel Spurr; at the Institute of Cancer Research-C. Strong, B. Smith and K. Chin (for technical assistance), and B. Ponder (for support and encouragement); and at CEPH-we acknowledge the ongoing invaluable support and encouragement of Jean Dausset and Daniel Cohen, expert computer-generated artwork by Dustin Cartwright and Peter Cartwright, patient preparation of the manuscript by Elizabeth May, and dedication and commitment of the entire technical staff to supporting construction of the human genetic map. The following grant support is acknowledged: The Medical Research Council of Great Britain and the Cancer Research Campaign (to the Galton Laboratory and Institute of Cancer Research); NIH Grant NS20012 (to J.G., Neurogenetics Laboratory, Massachusetts General Hospital); NIH Grant l-ROI-GM40543 (to H.D.-K., Department of Genetics, Washington University School of Medicine); the Lucille P. Markey Charitable Trust (to P.G., Department of Genetics, Washington University School of Medicine); and Institut National de la Sante et de la Recherche Medicale, INSERM (to H.C. and M.L., CEPH). REFERENCES 1. BOWDEN, D. W., GRAVIUS, T. C., GREEN, P., FALLS, K., WURSTER-HILL, D., NOLL, W., MULLER-KAHLE, H., AND DONISKELLER, H. (1989). A genetic linkage mapof 32 loci on human chromosome 10. Gerwmics 5: 718-726. 2. DAUSSET, J., CANN, H., COHEN, D., LATHROP, M., LALOUEL, J-M., AND WHITE, R. (1990). Centre d’Etude du Polymorphisme Humain (CEPH): Collaborative genetic mapping of the human genome. Genomics 6: 575-577. 3. DONIS-KELLER, H., GREEN, P., HELMS, C., CARTINHOUR, S., WEIFFENBACH, B., STEPHENS, K., KEITH, T., BOWDEN, D., SMITH, D., LANDER, E., BOTSTEIN, D., AKOTS, G., REDIKER, K., G~~vrus, T., BROWN, V., RISING, M., PARKER, C., POWERS, J., WA?T, D., KAUFFMANN, E., BRICKER, A., PHIPPS, P., MULLER-KAHLE, H., FULTON, T., NG, S., SCHUMM, J., BRAMAN, J., KNOWLMN, R., BARKER, D., CROOKS,S., LINCOLN, S., DALY, M., AND ABRAHAMSON, J. (1987). A genetic linkage map of the human genome. Cell 51: 319-337. 4. FARRER, L. A., CASTIGLIONE, C. M., KIDD, J. R., MYERS, S., CARSON,N., SIMPSON, N. E., AND KIDD, K. K. (1988). A linkage group of five DNA markers on human chromosome 10. Genomics 3: 72-77. 5. LANDER, E. S., GREEN, P., ABRAHAMSON, J., BARLOW, A., DALY, M. J., LINCOLN, S. E., AM) NEWBURG, L. (1987). MAPMAKER: An interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1: 174-181. 6. LATHROP, G. M., LALOUEL, J-M., JULIER, C., AND On, J. (1985). Multilocus linkage analysis in humans: Detection of linkage and estimation of recombination. Amer. J. Hum. Genet. 37: 482-498.
7. LATHROP, G. M., NAKAMURA, Y., CARTWRIGHT, P., O’CONNELL, P., LEPPERT, M., JONES, C., TATEISHI, H., BRAGG, T., LAU)UEL,
ET
AL.
J-M., AND WHITE, R. (1988). A primary genetic map of markers for human chromosome 10. Genomics 2: 157-164. 8. MATHEW, C., CHIN, K., EASTON, D., THORPE, K., CARTER, C., LIOU, G., FONG, S-L., BRIDGES, C., HAAK, H., KRUSEMAN, A., SCHI~R, S., HANSEN, H., TELENIUS, H., TELENIUS-BERG, M., AND PONDER, B. (1987). A linked genetic marker for multiple endocrine neoplasia type 2A on chromosome 10. Nature (London) 328: 527-528. 9. MORTON, N. E., LINDSTEN, J., ISELIUS, L., AND YEE, S. (1982). Data and theory for a revised chiasma map of man. Hum. Genet. 62: 266-270. 10. MORTON, N. E., MACLEAN, C. J., AND LEW, R. (1985). Tests of hypotheses on recombination frequencies. Genet. Res. 45: 279-286.
11. MORTON, N. E., AND ANDREWS, V. (1989). MAP, an expert system for multiple pairwise linkage analysis. Ann. Hum. Genet. 53: 263-269. 12. NAKAMURA, Y., LATHROP, M., BRAGG, T., LEPPERT, M., O’CONNELL, P., JONES, C., LALOUEL, J-M., AND WHITE, R. (19888). An extended genetic linkage map of markers for chromosome 10. Genomics 3: 389-392. 13. NAKAMURA, Y., LATHROP, M., O’CONNELL, P., LEPPERT, M., BARKER, D., WRIGHT, E., SKOLNICK, M., KONDOLEON, S., Lrrr, M., LALOUEL, J-M., AND WHITE, R. (1988b). A mapped set of DNA markers for human chromosome 17. Genomics 2: 302309. 14. NAKAMURA, Y., LATHROP, M., O’CONNELL, P., LEPPERT, M., LALOUEL, J-M., AND WHITE, R. (1988c). A primary mapof ten DNA markers and two serological markers for human chromosome 19. Gerwmics 3: 67-71. 15. NAKAMURA, Y., MATHEW, C. G. P., SOBOL, H., EASTON, D. F., TELENIUS, H., BRAGG, T., CHIN, K., CLARK, J., JONES, C., LENOIR, G. M., WHITE, R., AND PONDER, B. A. J. (1989). Linked markers flanking the gene for multiple endocrine neoplasia type 2A. Genomics 6: 199-203. 16. O’CONNELL, P., LATHROP, G. M., LAW, M., LEPPERT, M., NAKAMURA, Y., HOFF, M., KUMLIN, E., THOMAS, W., ELSNER, T., BALLARD, L., GOODMAN, P., AZEN, E., SADLER, J. E., CAI, G. Y., LALOUEL, J-M., AND WHITE, R. (1987). A primary genetic linkage map for human chromosome 12. Genomics 1: 93-102. 17. Paris Conference (1971). “Standardization in Human Cytogenetics. Birth Defects: Original Article Series,” Vol. 8, p. 7, The National Foundation, New York. 18. REEDERS, S., KEITH, T., GREEN, P., GERMINO, G., BARTON, N., LEHMANN, O., BROWN, V., PHIPPS, P., MORGAN, J., BEAR, J., AND PARFREY, P. (1988). Regional localisation of the autosomal dominant polycystic kidney disease locus. Genomics 3: 150-155. 19. SIMPSON, N., KIDD, K., GOODFELLOW, P., MCDERMID, H., MYERS, S., KIDD, J., JACKSON, C., DUNCAN, A., FARRER, L., BRASCH, K., CASTIGLIONE, GREENBERG, C., GUSELLA,
C., GENEL, J., HOLDEN,
M., GERTNER, J., AND WHITE,
J., B.
(1987). Assignment of multiple endocrine neoplasia type 2A to chromosome 10 by linkage. Nature (London) 328: 528-530. 20. SMITH, M., AND SIMPSON, N. E. (1989). Report of the committee on the genetic constitution of chromosomes 9 and 10, Human Gene Mapping 10 (1989), Tenth International Workshop on Human Gene Mapping. Cytogenet. Cell Genet. 61: 202-225. 21. TROFA?TER, J. A., GAINES, J. L., AND CONNEALLY, P. M. (1986). LIPIN: An interactive data entry and management program for LIPED. Am. J. Hum. Genet. 39: 147-148. 22. WARNICH, L., DIETZSCH, E., HERBERT, J., BRUSNICKY, J., LAUBSCHER, L., AND RETIEF, A. (1989). An anonymous DNA probe (H38) [DlOS37] detects a MspI polymorphism on chromosome 10. Nucleic Acids Res. 17: 474.
6,3%!-412
(1990)
The CEPH’ Consortium Primary Linkage Map of Human Chromosome 10 RAYMOND L. WHITE,* JEAN-MARC LALOUEL,* YUSUKE NAKAMURA,* HELEN DONIS-KELLER,t’* PHILIP GREEN,?* DONALD W. BOWDEN,* CHRISTOPHER G. P. MATHEW,~ DOUGLAS F. EASTON,~ ELIZABETH B. ROBSON,~~ NEWTON E. MORTON,B JAMES F: GUSELLA,# JONATHAN L. HAINES,# ANDRIES E. RETIEF,** KENNETH K. KIDD, t t JEFFREY C. MURRAY,*+ G. MARK LATHROP,*** AND HOWARD M. CANN***,* *Howard Hughes Medical Institute and Department of Human Genetics, University of Utah Medical Center, Salt Lake City, Utah 84 732; t Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63 110; *Collaborative Research, Inc., Bedford, Massachusetts 01730; glnstitute of Cancer Research, Sutton, Surrey SM2 SNG, England; “The Galton Laboratory, University College London, London NW1 2HE, England; YDepartment of Community Medicine, University of Southampton, Southampton SO9 4x/, England; #Neurogenetics Laboratory, Massachusetts General Hospital, Boston, Massachusetts 02114; **Department of Human Genetics, Faculty of Medicine, University of Stellenbosch, Tygerberg, 7505 South Africa; t t Department of Human Genetics, Yale University School of Medicine, New Haven, Connecticut 065 1o-8005; **Department of Pediatrics, Division of Medical Genetics, College of Medicine, University of Iowa, Iowa City, Iowa 52242; and ***Centre d’ftude du Polymorphisme Humain (CEPH), 75010 Paris, France Received
July 6, 1989;
revised
6, 1989
laborative effort, that of the CEPH collaboration to map the human genome (Dausset et al., 1990). The map was constructed from genotypic data generated from DNA of the 40 large nuclear families/pedigrees in the CEPH reference panel with probe and restriction enzyme combinations used by various laboratories participating in the collaboration. One laboratory provided data for the map from DNA of the CEPH reference panel plus 19 additional families. We also provide details of how the consortium map was prepared and analyzed. Human chromosome 10 has been described, cytogenetically, as a medium-sized, submetacentric chromosome, accounting for approximately 4.6% of the measured length of all the autosomes (Paris Conference, 1971). The mean genetic length of this chromosome in males, taken from an analysis of chiasma counts (Morton et al., 1982), is 127 CM. Linkage maps for chromosome 10, based on genotypes generated from the CEPH panel DNAs (or from other families) with probe and enzyme combinations from single laboratories, have already been published (Donis-Keller et al., 1987; Farrer et al., 1988; Lathrop et al., 1988, Nakamura et al., 1988a; Bowden et al., 1989). Recently the locus for a gene determining the disorder multiple endocrine neoplasia 2A (MENPA) has been assigned to chromosome 10 by linkage to already localized DNA polymorphisms (Mathew et al., 1987; Simpson et al., 1987). Genetic markers that have been used to improve
The first CEPH consortium map, that of chromosome 10, is presented. This primary linkage map contains 26 continuously linked loci defined by genotypes generated from CEPH family DNAs with 3’7 probe and enzyme combinations. C-genetic localization of some of the genetic markers indicates that the consortium map extends, at least, from lop13 to lOq26. The order of loci on the consortium map agrees with the physical localization data. The female map spans 309 CM (206 CM if an approximation of interference is included in the mapping function used to construct the map), and the mean genetic distance of intervals is 11 CM (7 CM). Also presented are maps of chromosome 10 from each of five CEPH collaborating laboratories, based on genotypes for all relevant markers in the CEPH database. The CEPH consortium map of chromosome 10 should be useful for localization of any gene of interest falling within the span covered. The genotypes in the chromosome 10 consortium map database are now available to the scientific community. o 1990 Academic Press,
November
Inc.
INTRODUCTION
We present here the CEPH consortium linkage map of human chromosome 10. This map represents a col’ CEPH, Centre d’Etude du Polymorphisme Humain. 2 To whom correspondence should be addressed at Centre d’Etude du Polymorphisme Humain (CEPH), 27 rue Juliette Dodu, 75010 Paris, France. 393
All
..
Copyright 0 1990 . _ . rlghta of reproduct!on
OSSEL7543/90 $3.00 by Academic Press, Inc. m any form reserved.
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the precision of localization of the MEN2A gene (Nakamura et al., 1989), enhancing prospects for diagnosis and isolation, are included in the CEPH consortium map. The localization on the consortium map of other markers closely linked to this gene will further refine its location. The CEPH consortium map also provides opportunities for localizing other genesof interest that reside on chromosome 10. MATERIALS
AND
METHODS
Genotypic Data Genotypes for 57 DNA polymorphisms, localized or possibly localized to chromosome 10, were generated from the CEPH,reference panel DNAs with probe and enzyme combinations used in nine CEPH collaborating laboratories. These 57 DNA polymorphisms represent 43 loci (Table 1). Genotypes for a subset of 27 of these 57 polymorphisms were generated in the CEPH panel plus 19 additional families by one of the collaborating laboratories (Nakamura et aZ., 1988a). Genotypic data were sent in standard format on 5.25-in. IBM-PC compatible floppy disks to CEPH, where they were incorporated into a chromosome 10 database suitable for preparing appropriate parameter and pedigree files for linkage analysis and mapping. All of the genotypic data have been examined for parental exclusions. In most of the participating laboratories, genotypes are checked by comparing them with the original autoradiograms, routinely, for multiple recombinants over relatively small genetic distances and/or for marker pairs showing less than 15% recombination. Table 1 summarizes the 57 probe and enzyme combinations for which there are genotypic data in the chromosome 10 database. Eleven of these DNA polymorphisms possessfour or more alleles. Two or more probe and enzyme combinations detected RFLPs at each of five loci, RBP3, DlOSll, DlOS16, DlOS34, and DlOS43. It should be noted that probe cTBIRBP9 (system 23 in Table l), isolated from a human genomic cosmid library with a cDNA probe for RBP3 (Nakamura et al., 1988a), generated genotypes that showed recombination with those of three other RBP3 markers, CRI-CS762/HincII (recombinant individuals 134509, 135007, and 140806), CRI-CTlS/MspI (recombinant individual 135007), and CRI-CS768/TaqI (recombinant individuals 134507 and 134509). These genotypes have been confirmed on the original autoradiograms, and there is no evidence for misidentification or contamination of the DNA from these individuals at CEPH. Although some of the DNA polymorphisms listed in Table 1 have been localized to chromosome 10 by physical mapping, techniques, others have been assigned to this chromosome by linkage to one or more previously assigned markers. Note that 5 polymor-
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phisms (representing four loci) listed in Table 1 are not localized to chromosome 10 (systems 32, 35, 55, 56, and 57). When the chromosome 10 database was prepared, these 5 markers were considered to be assigned or possibly assigned to this chromosome. Subsequently, analyses performed by laboratories participating in the construction of the consortium map failed to demonstrate linkage of only these 5 markers with the other 52 markers in the chromosome 10 database. Consistent with this finding, assignment of these 5 markers to other chromosomes has been reported. Thus, of the 57 DNA polymorphisms in the chromosome 10 database, 52, representing 39 loci, are assigned to this chromosome. For a number of probe and enzyme combinations, the observed heterozygote frequencies presented in Table 1 are based on all parents tested in the laboratories rather than the parental genotypes found in the database. Some laboratories contributed genotypic data to the database for only informative families (i.e., at least one heterozygous parent), although parents of uninformative families had been tested. The last column in Table 1 indicates how many individuals of the CEPH reference family panel were genotyped for each probe and enzyme combination. The CEPH panel consists of 517 individuals who account for 530 positions in the 40 reference families, because some individuals appear in more than 1 family. Those probe and enzyme combinations typed in more than 530 individuals were used in one of the collaborating laboratories with the CEPH panel and 19 additional families. Eight probe and enzyme combinations were each tested in lessthan 200 individuals, and six of these detected polymorphisms in the complex locus RBP3 or DlOSll. Two probe and enzyme combinations, H4/MspI and H4/BgZII, were used to genotype CEPH families by more than one laboratory. Two hundred fifty-one individuals were typed in duplicate for H4/MspI by different laboratories, and two individuals showed discrepant genotypes. Discrepant results were found for a single individual in a set of 109 CEPH panel members who were typed twice for H4/BgZII by different laboratories. The frequency of discrepant genotypes approximates 1% for these systems. These discrepant genotypes were not included in the chromosome 10 database. Correction of Genotypes Each contributor of genotypes was sent a chromosome 10 database. After distribution, five genotypes were corrected in the database as follows: The genotypes for CRI-L647 (DlOSll) of CEPH panel individual 134411 and for CRI-CS931 (DlOSll) of individuals 134405 and 134411 were not used for construction of maps. The genotype for CRI-JlOl/HindIII (DlOS43)
CEPH
CONSORTIUM
LINKAGE
TABLE Summary
of CEPH
MAP
OF
CHROMOSOME
395
10
1
Chromosome
10 Consortium
Markers
System”
Probe
Enzyme
Markep
Localization
Number of alleles”
Heter.d
Number typed
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57
ILPR pTHHlOB-1 pYNZ156 pTHH54 pl-101 OS-2 pTBlO-171 pMCK2 pMHZ15 H4 H4 pCMM17-1 pCMM17-4 pTBlO-163 cTBQ4 cTBQ7 cTBQ-12 cTBQ16 cTBQ20 pMCT122-2 pEFD75 pEFD70-2 cTBIRBP9 cTB14-15 cTB14-16 lcTB1434 2cTB14-34 cTB1436 p9-12a zap32 CDCP EMBL3-93 hp4f 1 cmhl0-2 pL28-5 HOATl-5 H38 CRI-L368 CRI-L647 CRI-JM14 CRI-JlOl CRI-JlOl CRI-CS762 CRI-J125 CRI-J127 CRI-CS931 CRI-CS932 CRICS933 CRI-CT19 CRI-CS933 CRICS762 CRI-CS768 CRI-J170 VTR4 H31 M7 M7
L?gZI B&II MspI MspI %I Hind111 PVUII RsaI MspI B&II MspI MspI PVUII MspI MspI TaqI Td MspI MspI TuqI TuqI PVUII TuqI TaqI RsaI TaqI ToqI MspI %I Hind111 TaqI MspI BclI B&II XmnI MspI MspI Hind111 TaqI Hind111 HindIII EcoRI HincII HindIII H&III TaqI Hind111 BamHI MspI TaqI TW TaqI PstI EcoRI HindIII BamHI MspI
ILPR DlOS13 DlOS18 DlOS14 DlOS4 DlOS20 DlOS19 DlOS15 DlOS17 RBP3 RBP3 DlOS16 DlOS16 D10S22 DlOS27 DlOS28 DlOS29 DlOS30 DlOS31 DlOS36 DlOS25 DlOS26 RBP3 DlOS32 DlOS33 DlOS34 DlOS34 DlOS35 DlOS5 EGR2 CDCP D9S37 VIM DlOS39 D13S62 OAT DlOS37 DlOS12 DlOSll DlOS62 DlOS43 DlOS43 RBP3 DlOS44 DlOS45 DlOSll DlOSll DlOSll RBP3 DlOSll RBP3 RBP3 DlOS49 DlOS6 DllS348 D7S422 D7S422
lOp15-p14 10 10 10 lOq22-q23 10 lOq21.1-q22 10 lOpter-p13 10qll.2 lOq11.2 10 10 lOq21.1 10 lOpter-p13 10 10 10 10 10 10 lOq11.2 10 10 lOpter-ten lOpter-ten 10 lOq21.1 lOq21.1 lOq21.1 9 lop13 1Optercen 13 lOq26 lOq25.1 10 lOq11.2 10 10 10 lOq11.2 10 10 lOq11.2 lOq11.2 lOq11.2 lOq11.2 lOq11.2 lOq11.2 lOq11.2 10 lOq26 llq21-qter 7p11.2-q21.1 7p11.2-q21.1
2 2 2 3 3 3 2 7 2 2 2 2 2 3 2 8 2 2 2 2 6 4 2 3 5 2 2 2 2 2 2 2 2 2 2 2 2 9 5 3 2 2 2 2 2 2 2 5 9 6 2 2 3 9 2 2 2
0.09 0.38 0.26 0.62 0.54 0.61 0.48 0.30 0.52 0.20 0.32 0.41 0.31 0.58 0.50 0.96 0.48 0.46 0.45 0.27 0.60 0.52 0.41 0.64 0.80 0.49 0.46 0.55 0.39 0.10 0.42 0.35 0.41 0.32 0.32 0.35 0.45 0.66 0.50 0.63 0.21 0.38 0.30 0.45 0.31 0.44 0.43 0.26 0.26 0.33 0.33 0.21 0.59 0.63 0.37 0.45 0.36
456 523 480 556 594 572 725 732 516 532 721 689 699 612 722 488 640 557 663 719 603 631 604 524 383 581 575 510 343 162 326 342 300 186 422 355 321 293 214 430 210 224 282 325 301 180 373 92 137 135 25 87 348 333 302 371 357
’ Sequential database numbers of probe and enzyme combinations. * Official Human Gene Mapping Workshop symbols. ’ Number of alleles for the polymorphism detected by the probe and enzyme d Observed heteroxygote frequency (see text, Materials and Methods).
combination.
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of individual 1204 was changed from 12 to 22 and that for CDC2 of individual 142404 was corrected to 12 from 11. Linkage
Maps from Collaborating
Laboratories
Five laboratories prepared maps with genotypes from the chromosome 10 database using programs for multilocus linkage analysis, CRI-MAP (Donis-Keller et al., 1987), LINKAGE (Lathrop et al., 1985), or MAPMAKER (Lander et al., 1987), and/or for multiple pairwise linkage analysis, MAP (Morton and Andrews, 1989). Six maps were prepared by the five laboratories. These maps were circulated among all contributors of chromosome 10 genotypes (members of the consortium map committee),*and they were compared for locus order, statistical support of orders, genetic distances of intervals, and sex differences in recombination frequencies. The committee met to analyze and discuss these comparisons and plan the final consortium map. RESULTS
AND
DISCUSSION
The Consortium Map of Chromosome 10 Preliminary analysis showed that markers H31/ HindIII, M7/BamHI, M7/MspI, EMBL3-93/MspI, and pL28-5/XmnI (see Table 1) could not be assigned to chromosome 10 by linkage. Subsequently, H31 and M7 were reported to be assigned to chromosomes 11 and 7, respectively, by in situ hybridization (A. Retief, personal communication); they are now designated DllS348 and D7S422. The loci defined by EMBL3-93 and pL28-5 were reassigned to chromosomes 9 and 13, respectively, by analysis of human-rodent somatic cell hybrid cell lines; these loci are designated D9S37 and D13S62. With the exclusion of these five markers, there remain in the database genotypes for 52 probe and enzyme combinations, representing 39 loci, assigned to chromosome 10. These genotypes are the actual basis for the construction of the six chromosome 10 maps contributed by five collaborating laboratories. These maps are presented below (see Chromosome 10 Maps from Individual Laboratories). The chromosome 10 linkage maps from collaborating laboratories were quite useful for the preparation of the consortium map. The maps allowed us to assemble a subset of genetic markers likely to be placed uniquely on the consortium map. This subset consisted of 29 chromosome 10 loci (41 probe and enzyme combinations), most of which were localized on at least four individual maps with acceptable support. The genotypes for this subset of loci contained the corrections listed above for DlOSll and DlOS43 (see Materials and Methods); locus CDCP was not included in the subset. RBP3 was included in this subset; it was defined by a haplotype constructed from genotypes for seven
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probe and enzyme combinations including the original genotypes for cTBIRBPS/TaqI. Members of the consortium map committee were asked to determine the maximum likelihood order of those of the 29 loci that could be assigned unambiguously (odds of 1OOO:l or greater) on the map. All of the 29 loci could not be localized unambiguously on a map, and the order containing the most loci to be localized was accepted as a tentative chromosome 10 consortium map. This order contained 26 loci. The placement of DlOSll, one of the 26 loci on the tentative consortium map, is problematic. DlOSll could not be localized with a desired support value in five of the individual maps constructed, based on genotypes of all or many chromosome 10 loci in the database (seebelow, Chromosome 10 Maps from Individual Laboratories). For the localization of DlOSll on the tentative consortium map, the level of support depended critically upon the specific subset of loci used in the analysis. When genotypes for all of the remaining tentative consortium map loci were included, the odds for placing DlOSll decreased substantially. The decrease in support may reflect errors in the genotypic data for these additional markers, causing inconsistencies in the dataset being analyzed and leading to lower likelihood support values. As the odds favoring the given location on the consortium map for DlOSll appear to depend on which markers were used for the analysis, the localization is open to question. Accordingly, DlOSll is not included on the consortium map. Three additional loci (4 probe and enzyme combinations) were placed on the tentative consortium map, DlOS15, DlOS17, and DlOS43. Pairwise lod score and multilocus linkage analyses indicated that each of these loci did not recombine with a locus assigned to the map (RBP3, DlOS39, and DlOS12, respectively). Furthermore, each locus could not be localized with the usual support to any other position on the tentative consortium map. A distance of 2.7 CM for the interval between DlOS17 and DlOS39 is reported for the male map from one individual laboratory (Fig. 4). However, this map is only slightly more probable (by odds of 1.3:1) than one with no recombination between these 2 adjacent loci for both sexes (see discussion under Chromosome 10 Map from the Institute of Cancer Research, below). Furthermore, this map was constructed with all 39 chromosome 10 loci, while DlOS17 was added to the tentative consortium map containing a subset of loci from the database. Addition or exclusion of loci in multipoint map construction could account for the difference between the consortium map and that from the collaborating laboratory for the genetic distance of this interval. With the addition of DlOS15, DlOS17, and DlOS43 and the exclusion of DlOSll, the consortium map contains 28 loci (37 probe and enzyme combina-
CEPH
CONSORTIUM
LINKAGE
tions). The order of these 28 loci is shown in Fig. 1, the chromosome 10 consortium map. The consortium map order reflects near-unanimous committee agreement. For each of the 28 loci on this map, localization at the position indicated is agreed upon by at least four of the six maps from laboratories of representatives on the consortium map committee. Moreover, no locus omitted from the map is localized acceptably on more than three maps from the individual laboratories. With the component loci and their order on the consortium map established, sex-specific genetic distances of intervals were computed for the map by multilocus linkage analysis (no interference assumed) and multiple pairwise analysis (interference specified). The genotypic data used for these computations were corrected for a single genotype for marker DlOS43 (see Materials and Methods); other loci with corrected genotypes (DlOSll and CDCZ) were not included on the consortium map. Despite evidence for recombination with other RBP3 markers, cTBIRBP9 was considered a nonrecombining component of this complex locus for the distance computations. Both sets of sex-specific interval distances are presented in Table 2. The chromosome 10 consortium map contains 28 loci continuously linked from locus DlOS31 (inferred from the genetic data to reside on distal 1Op) to DlOS6 localized to lOq26 (Fig. 1). With multilocus linkage analysis, the mean interval distance for the female map, which spans 309 CM (Haldane), is 11 CM (Table 2). With multiple pairwise analysis using a mapping function that provides an estimate for interference, the female map spans 206 CM and the mean interval distance is 7 CM (Table 2). Table 2 shows that recombination frequencies are higher in females for at least 18 of the 27 intervals of the consortium map. Testing of the sex-factored pairwise lod scores for each interval on the map revealed a significant female excess in recombination frequencies for the interval DlOS49-DlOS34 (Xf = 6.80). Significant excesses of male recombination frequencies were found for intervals DlOS33-DlOS32 (X4 = 6.32), DlOS20-DlOS18 (x; = 7.76) (discussed in Nakamura et al., 1988a), and DlOS36-DlOS6 (XT = 7.55); a borderline excess was found for interval DlOS62-DlOS4 (x! = 4.02). Note that the first and third of these intervals with increased recombination in males are near and at the ends of the map, respectively, consistent with earlier observations for chromosomes 10, 12, 16, 17, and 19 (Nakamura et al., 1988a; O’Connell et al, 1987; Reeders et al., 1988; Nakamura et al., 1988b; Nakamura et al., 1988c). The sex difference for the most distal interval on 1Oq (DlOS36-DlOS6) is especially interesting, because it replicates the excess of male recombination frequency found in this region between DlOS36 and DlOS25 (Nakamura et al., 1988a).
MAP
OF
CHROMOSOME
10
397
Information concerning the physical localization of 17 loci in the chromosome 10 database, 12 of which are on the consortium map, is available (presented in Table 1, shown for selected loci in Fig. 1) (Bowden et cd., 1989; Smith and Simpson, 1989; Warnich et al., 1989). The order of the physical (cytological) locations agrees with the order of these loci on the consortium map. The comparison of physical and genetic orders here is not stringent because some of the physical locations overlap; e.g., that of DlOS34 could be anywhere on 1Op and that of DlOS19, localized to lOq21.1-q22.0, overlaps with those for DlOS22, DlOS5, and DlOS4. If one omits DlOS34 and DlOS19 from the comparison, physical and genetic orders still agree. As DlOS34 is localized to lop and RBP3 to lOq11.2, the centromere may be placed between these adjacent loci, in an interval of 8.7 CM or less. Loci RBP3 and DlOS5 have been shown to be linked to the locus of the gene determining MENBA (Mathew et al., 1987; Simpson et al., 1987). It has already been suggested that markers DlOS15, DlOS34, and DlOS30 (not included in the consortium map) will be useful for the localization of the MEN2A gene (Nakamura et al., 1988a). Indeed, recent evidence indicates that the MENZA gene lies between DlOS34 and RBP3/DlOS15, in the pericentromeric region (Nakamura et al., 1989). Consortium and individual maps confirm the significance of these markers and suggest that DlOSll should also be considered for improving the precision of localization of this disease-determining gene. This consortium map of 28 loci should be useful for primary localization of any gene of interest falling within the region of chromosome 10 covered. A subset of 15 marker loci, 14 from this map (DlOS31, DlOS33, DlOS35, DlOS28, VIM, DlOS49, RBPS, DlOS22, DlOS16, DlOS14, DlOS62, DlOS20, DlOS12, DlOS27, and DlOS6), chosen for informativeness (9 of the 15 loci possess three or more known alleles) and spacing along the map, should permit detection of linkage with most genes on chromosome 10 which determine inherited disorders, given sufficient meioses from appropriate families, e.g., those in which a disease-determining gene segregates. The basic strategy used to construct the consortium map involved ordering a subset of chromosome 10 loci likely to be uniquely localized with an acceptable level of support. Maps constructed with the chromosome 10 database by laboratory groups participating in the consortium map project enabled us to determine which loci were likely to be uniquely localized. The disadvantage of this strategy was the exclusion of 11 chromo‘Locus DlOS27 is not on the final consortium map, but it has been mapped on the preliminary maps. It is included in this subset of markers because it is located in the Bame region as and more informative than DlOS36.
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FEMALE
IlOS35
-
mS39Jms17 llOS28
15
MALE
14
DIOS31 13
D10S33
‘IM
12.3
D10S32
12.2 12.1
DlOS35
)lOS49
I 11.2
:
DlOS14 DlOS62
)lOS34
)lOS22 D10S29
>I0519 >I0516
DlOs14 22.1 22.2 22.3
l-4 1
1
DlOS29
D10S62
DlOS4 D10S36
23.1 23.2
D1OS1891omO
23.3
DICE+6 I
-
24.1 24.2
25.1
&QpMOs43
25.2 25.3
DlOS44
26.1
DlOS36 DIOS6
26.2 26.3
10
CEPH
CONSORTIUM
LINKAGE
some 10 loci from the consortium map. The alternative approach would have been to place on a map all of the 39 chromosome 10 loci at their most likely positions. In this case the drawback would be some regions with uncertain locus order. Both strategies have their merits, and it is useful to consider the reason(s) for constructing a genetic map before embracing or rejecting a particular approach. If the reason for constructing a primary map is to use it to localize genes of interest, unambiguous localization of the component markers is of paramount importance. The main motivation for organizing the CEPH collaboration to genetically map the human genome was (and still is) to use the resultant maps of chromosomes to localize genes of interest, essentially those that determine inherited diseases. This consideration, thus, underlies the strategy used to construct the CEPH consortium map of chromosome 10. Chromosome
10 Maps from Individual
Laboratories
Six maps of chromosome 10 contributed by five CEPH collaborating laboratories participating in the construction of the consortium map are presented here. The contributing groups were from the Howard Hughes Medical Institute at the University of Utah (laboratory 1); the Department of Genetics, Washington University School of Medicine (laboratory 2); the Galton Laboratory (laboratory 3); the Institute of Cancer Research (laboratory 4); and the Neurogenetics Laboratory, Massachusetts General Hospital (laboratory 5). These maps provided useful information, especially on locus order, which helped guide the construction of the CEPH consortium map of chromosome 10. Each of the maps was constructed from the data in the chromosome 10 database (containing genotypes for 57 probe and enzyme combinations representing 43 loci), and laboratories 2,3,4, and 5 used the corrections for five genotypes, as indicated above (see Materials and Methods), for the entire map construction process or in its final stages. Each laboratory used the original genotypes for marker cTBIRBP9 except for laboratory 3, which elected to avoid recombination within the RBP3 locus, as described under the section contributed by that group. When the five laboratory groups found that 4 loci (5 probe and enzyme combinations), detected by probes EMBL3-93, pL28-5, H31, and M7 (Table l), could not be linked to any of the other 39 loci in the chromosome 10 database and/or had been reassigned to other chromosomes, they excluded them from further consideration. The task for each group, then, was to prepare a map of the remaining 39 loci.
MAP
OF
CHROMOSOME
10
399
One or both of the basic strategies for map construction were used by each of the five laboratories. Laboratories 1, 2, and 5 constructed maps of chromosome 10 containing only unambiguously localized loci as determined by an acceptable support criterion for localization (Figs. 2,3, and 5). Laboratories 2,3, and 4 constructed maps with all of the 39 chromosome 10 loci placed at their most likely positions and used measures of local support to test for order (Fig. 4 and Tables 3, 4, and 5). Each of the five laboratories constructed a map with one of three programs for multilocus linkage analysis. Laboratory 3 also used the MAP program for multiple pairwise analysis to construct a map, building a trial map from data in lod score tables for all possible pairs of chromosome 10 loci. This map was then refined until all loci were at their most likely positions (described below under Chromosome 10 Map from the Galton Laboratory). The multilocus linkage analysis programs sequentially incorporate loci into a starting map. In each case, loci were incorporated, one by one, into a starting map, on the basis of a previous publication (laboratory 1) or consisting of an initial pair (laboratories 2 and 4) or larger subset (laboratories 3 and 5) of loci. A likelihood criterion is used to distinguish possible placements of the locus being added to the map. In general, the laboratories used an odds ratio of 1OOO:l or greater to uniquely localize a marker on the map and to distinguish between two orders of loci. Laboratory 1 found very stringent criteria of support for loci on the initial map (see Fig. 2) and localized additional loci with a minimal odds ratio of 1OO:l. Additional (local) support for a given order was computed by some of the laboratories as the likelihood difference(s) obtained from permuting the positions of two or more adjacent loci (see Fig. 2 and Table 3). For the maps constructed by laboratory 3, measures of local support were used to indicate uncertainty of order with respect to adjacent pairs of loci (Tables 4 and 5). Linkage maps can be depicted in various ways, as is evident from Figs. 2, 3, 4, and 5 and Tables 4 and 5. The maps consisting of uniquely localized loci indicate possible positions of ambiguously placed loci (Figures 2,3,4, and 5 and Table 6). Maps presenting an optimal order for the entire set of (39) chromosome 10 loci show support for permitted local permutations within that order (Tables 3, 4, and 5) or distinguish uniquely localized loci from the others (Fig. 4). Three maps present interval distances as recombination frequencies (no crossover interference assumed; Figs. 2 and 5) or cen-
FIG. 1. CEPH consortium linkage map of human chromosome 10. Two maps are shown, one based on female recombination frequencies and the other on male recombination frequencies. Interval distances shown on these maps are proportional to centimorgans. Loci shown on the same line are not separable by recombination. Recombination frequencies and the distances (centimorgans) for intervals are presented in Table 2. Physical localizations of selected map loci to regions on chromosome 10 are shown.
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TABLE Recombination
Frequencies Multilocus
(0) and Centimorgans:
DlOS31 DlOS33 DlOS32 DlOS35 DlOS39/DlOS17’ DlOS28 VIM DlOS49 DlOS34 RBP3/DlOS15’ DlOS5 DlOS22 DlOS19 DlOS16 DlOS14 DlOS29 DlOS62 DlOS4 DlOS20 DlOS18 D10S12/D10S43’ OAT DlOS44 DlOS36 DlOS6 Total
Chromosome
10 Consortium
analysis
Multiple
e Locus
2
Female
Male
Female
0.09 0.03 0.16 0.09 0.08 0.18 0.20 0.15 0.08 0.08 0.11 0.08 0.14 0.12 0.08 0.11 0.07 0.14 0.00 0.30 0.04 0.06 0.17 0.02
0.08 0.12 0.14 0.06 0.05 0.09 0.05 0.05 0.02 0.05 0.00 0.08 0.08 0.11 0.02 0.03 0.12 0.09 0.16 0.06 0.07 0.00 0.13 0.22
9.9 3.1 19.3 9.9 8.7 22.3 25.5 17.8 8.7 8.7 12.4 8.7 16.4 13.7 8.7 12.4 7.5 16.4 0.0 45.8 4.2 6.4 20.8 2.0
8.7 13.7 16.4 6.4 5.3 9.9 5.3 5.3 2.0 5.3 0.0 8.7 8.7 12.4 2.0 3.1 13.7 9.9 19.3 6.4 7.5 0.0 15.1 29.0
309.3
214.1
n Centimorgans computed from recombination frequencies b See Refs. (10) and (11). ’ Loci separated by / were not separable by recombination.
Male
by the mapping
timorgans computed from recombination frequencies (no interference) by the Kosambi mapping function which partially corrects for the effects of interference (Fig. 4). The interval distances are cumulated, starting from the most terminal pter marker, for the maps in Tables 4 and 5, these distances being, respectively, centimorgans computed with the Kosambi function and those derived from recombination frequencies estimated with a mapping function which specifies interference. Accordingly, the intervals and total length for the map presented in Table 5 tend to be shorter than those for the other maps. Figure 3 presents a map of uniquely ordered loci, with possible placements for other loci, but does not specify interval distances. As there is evidence for variation in recombination frequencies by sex, the maps in Figs. 4,and 5 and Tables 4 and 5 present sex-specific genetic distances. Chromosome 10 Map from the Howard Hughes Medical Institute, University of Utah. The 57 genetic systems available were reduced to 45 loci after haplotypes
analysis
eb
CM”
distance
pairwise
Map
function
CM*
Female
Male
Female
0.07 0.02 0.12 0.06 0.06 0.14 0.13 0.12 0.06 0.07 0.08 0.07 0.07 0.15 0.05 0.08 0.07 0.10 0.03 0.20 0.01 0.08 0.14 0.03
0.06 0.11 0.08 0.05 0.05 0.03 0.05 0.03 0.00 0.03 0.02 0.05 0.06 0.05 0.02 0.01 0.11 0.06 0.08 0.08 0.06 0.00 0.11 0.18
6.8 2.5 12.8 6.2 6.0 14.6 13.6 11.7 5.7 7.2 8.1 6.9 7.4 15.3 5.5 7.9 6.8 10.6 2.9 21.4 1.1 8.4 14.1 2.7 206.1
Male 6.1 11.0 8.1 5.1 4.8 3.1 4.8 3.1 0.0 3.1 1.7 5.5 6.0 4.6 1.8 1.5 10.7 6.3 7.9 8.4 5.6 0.0 11.4 18.9 139.7
of Haldane.
were constructed, defining the complex systems DlOS16 (probe and enzyme combinations 12, 13), DlOS34 (26, 27), DlOS43 (41, 42), RBP3 (10, 11, 43, 49, 51, 52), and DlOSll (39, 46, 47, 48, 50) (see Table 1). System 23 (CTBIRBPS), which shows recombination with other markers that also define RBP3, was not included in the haplotype for this complex system. Further reduction was obtained by deleting five systems (32, 35, 55, 56, 57) for which we could not find significant linkage to other systems. To construct a continuous map of chromosome 10, we elected to take advantage of a previously established primary map (Nakamura et al., 1988a). Because these loci originated from our laboratory, the original autoradiograms were available for data checking; moreover, most of these markers were tested on an expanded reference panel, and they appeared to span most of the chromosome. This map included 21 loci. Although most loci were suitably spaced and ordered with well-supported likelihoods, a few clusters of closely linked
CEPH
CONSORTIUM
LINKAGE
MAP
OF
CHROMOSOME
401
10
Initial Map 23 075
57 075 I
89 143 I
16 105 I
164 274 I
41 121 I
105 ooooomoooooww
45 082
I
70 095
I
77 128
85 183
I
125 190 11111111:1111111111111111
I
18 108
I
16 114
I
42 271
I
16 154
I
17 180
I
055 104 11111111:1111111111111111
DlOSll
L647
,
, TBIRBP9
RBP3
, TBQ16
DlOS30
271 owooooooooooowooooooooooowoooo 230 33333333333333333333333:333333333 128 oooooO0oooooOW
I
183 000000000000000
174 081 4U444444:4au44uu4~
100
I
I
$5bb '+a , I CDCZ
J127
rap32
DlOS45
DlOS62
JM14
, L EFD70
EGR2
,
,
H36
DlOS26
DlOS37
FIG. 2. Chromosome 10 map from the Howard Hughes Medical Institute, University of Utah. Initial map: The top row of figures represents 2 In likelihood support of order shown on the map for adjacent pairs of loci versus the inverted order. The second row of figures presents the recombination frequencies (X10’) for the intervals on the map, estimated under the assumption of no sex differences for recombination frequencies. Final map: Odds for the position shown for each locus on the map are $009 or greater than those for any other position. Each locus that could not be placed on the map with this criterion of support is shown under the map at its most likely position. Reiterated digits indicate the iteration during which a previously unlocalized locus was positioned on the map (e.g., 2222222 indicates the second iteration). Recombination frequencies shown above reiterated digits are those estimated for those map intervals receiving previously unlocalized loci during the indicated iteration. The full DlOSll haplotype is indicated here by the index probe, (CRI-)L647.
markers presented ambiguities. For each such cluster, we selected the most informative marker. Thus 16 anchor loci were available and strong support for their reported order was confirmed. There remained 24 loci to place on the map. Because the addition to a map of loci for which only poor support can be achieved complicates map construction inordinately, further mapping was performed with the program LINKAGE (Lathrop et al., 1985) through the following iterative algorithm. At a given iteration, the loci consist of two sets, a set of mapped loci and a set of as yet unmapped loci. The mapped loci define elementary intervals with estimated recombination values and documented support. Addition of loci to this map proceeds through the following steps.
(1) Regional assignment: For each unmapped locus, we find the subset of elementary intervals where this locus might lie by computing location scores on the current map; the candidate region may have already been reduced at previous iterations. (2) Selection of candidate anchors: In each interval, we select the locus that has the greatest odds of belonging in that interval, with some condition on minimum support. (3) Validation: For each candidate anchor, we validate the order of flanking loci and retest likelihood for location in other intervals within certain odds by maximum likelihood estimation; when candidates fall in adjacent intervals, further validation is performed using candidate anchors jointly.
24
28
9
33
53
26
30
-
-
-
-
45
-
-
12
7-----------------------~-
30
7--------------------~-
73~-----------------~30
-
-
-
31
-
-
4
-
1245-----------------
-451230--------------
123045-----------------
-4530------------------
-3031------------------
31
30
-
-
_
7
-
_
14
-
-
-
-
-
2
-
-
40
-
-
5
-
~
6
-
-
3
--3637--------
2-------------
40
217--------------
17
-
-
37
-42
42
37
-
_
38
36-
37
36-----
-
-
42
Note. Locus orders that differ from the best order by permuting some successive triple of loci and that have relative odds (as indicated best order, of at least 1:lOOO. Numbering of loci is as in Table 1; haplotyped systems are represented by the index of one of the component
---------~---------------------------2122
--------------------_______________
-----------------------------
36
42
-
-
-
29
-
-
39
----------------------------
11-
8
-
*1*-----------------------__
-
11
26
8
--------------------________
18
11
-42-37-----
-
18
with Relative Odds of within lOOO-fold of Best Order: Chromosome from the Department of Genetics, Washington University
3
---------------~------------
-------------------____
--------------------
------------------
---------_-----____
------------_____
16
Orders
g-----------------------~~---~~~~
34
1---------~--------------------------
25
1----------~-----------------~~~---~~~
19
-------------___
-----34
19
1925
1
Locus
TABLE
-
-
-
36
-
_
-
15
21 22
-
-
--_
_
_
54
-
_
_
20
21
-
-
-
-
-
_
_
22
to the right of the row), probes (Table 1).
-
----
-
-
44
10 Map
with
54
54
-
-
-
-
~
-
21
respect
to the
1:50
1:79
1:4.4
1:l
1:l
I:1
1:l
1:l
1:740
1:54
1:40
1:66
1:340
1:2.7
1:l
1:830
1:17
1:830
1:620
1:27
P1.2
1:l
1:l
1:130
1:l
z P r
8
CEPH
CONSORTIUM
LINKAGE
(4) Map update: The new anchors are added and recombination frequencies are reestimated in intervals receiving new anchors. This process is controlled by progressively decreasing the minimum support required before inclusion of new anchors, from minimum odds of 10,000 to 1000 and 100. All loci that could not be assigned to the map with odds greater than 100 were deliberately not included, as only their likely location can be trusted, rather, we summarized support for their location along the final map. All mapping was performed without considering sex differences in recombination and under the simplifying assumption of no interference. Our mapping process is summarized in Fig. 2. It should be noted that since this map was generated, the following loci have been subjected to some revision: DlOSll (systems 39 and 46), and DlOS43/HindIII (41) and CDC2 (31), as indicated above (see Materials and Methods). This may account for some minor differences with mapping results reported by other individual laboratories. Chromosome 10 Map from the Department of Genetics, Washington University School of Medicine. We first constructed an initial map by sequential incorporation of loci, using the “build” option in CRI-MAP (Donis-Keller et al., 1987). Loci were sorted in decreasing order of their number of informative meioses, and the first pair of loci in this list (DlOS28 and DlOS33) was chosen as the nucleus of the map. The next remaining locus was then placed in each possible position with respect to the nuclear map, and the multilocus likelihood was computed for each resulting locus order. Any order having a lOOO-fold lower likelihood than some other order for the same loci was excluded. If the locus was uniquely placed by this criterion it was incorporated into the map; otherwise, the next locus in turn was considered. In either case, a list of the permissible (nonexcluded) orders was appended to an orders database. Each time a locus was added to the map the entire process was repeated, placing loci with respect to the new map. At each stage, to reduce the number of likelihoods to be computed, only those possible locus placements that were compatible with the results of the prior tests stored in the orders database were considered. This procedure resulted in an ordered framework of loci whose order is unique, in the sense that any other order of this set of loci is inconsistent with the orders database; in other words, any other order is inconsistent with the results of some likelihood test-usually based on a subset, or an overlapping set of loci, rather than on the framework loci themselves-at 1OOO:l odds. (Since these tests may include loci not in the framework, this does not imply that all alternative orders for the framework loci will necessarily have 1000-fold lower likelihoods than the framework order.) In addi-
MAP
OF
CHROMOSOME
403
10
tion, the information in the orders database indicates the possible placements for the remaining loci with respect to the framework. The corresponding likelihood for each of these placements was computed (using the “instant” option of CRI-MAP). A schematic diagram summarizing the results of this analysis is shown in Fig. 3. Since this type of representation does not indicate the possible placements of ambiguously placed loci in the same region relative to each other, and since joint analysis of the genotypic information for such loci can
Dl OS31 &R
Dl OS33
I:170
DlOS32
I:01
DlOS35 Eos17
Dl OS39
1:1
DlOS26 VIM Dl OS49 Dl OS34 RBP3 DlOSll Dl OS5 Dl OS22 DlOSl9 DlOSl6 D10S14 I:59
D10S13
Dl OS29
I:1
Dl OS62 DlOS4 Dl OS20 DlOSl6 DlOSl2 OAT
3
1 J :t I:10
D10S27
I:56
Dl OS44
-I
1
Dl OS36 DlOS6
1
3 i&253
+;26
FIG. 3. Chromosome 10 map from the Department of Genetics, Washington University School of Medicine. The map, shown on the left, contains uniquely placed loci localized with odds of at least 1OOO:l. Possible placements, and their relative odds, of other chromosome 10 loci with respect to the map (the “best” placement is that with 1:l odds) are shown on the right. Other placements for these loci are excluded by odds of at least 1OOO:l.
404
WHITE
ET
AL.
TABLE Chromosome
10 Map
from
the Galton
Laboratory:
4 CRI-MAP
Analysis
of CEPH Location
Chromosome (CM)
System
Probe
Enzyme
Locus
Female
Male
1 19 25 24
IL2R cTBQ20 cTB14-16 cTB14-15 cTBl436 pMHZ15 cmhlO-2 cTBQ7 hp4f 1 CRI-J170 cTBQ16 DlOS34 pMCK2 RBP3 DlOSll p9-12a pTBlO-163 pTBlO-171 CDCP zap32 DlOS16 CRI-J127 pTHH54 cTBQ-12 pTHHlOh1 CRIJMl4 pl-101 OS-2 pYNZ156 CRI-L368 H38 CRI-JlOl HOATl-5 CRI-J125 cTBQ4 pMCT122-2 VTR4 pEFD70-2 pEFD75
B&I MspI RsaI TaqI MspI MspI BglII TagI BclI PstI MspI Haplo RsaI Haplo Haplo TaqI Ms.1 PULL11 TaqI Hind111 Haplo Hind111 MspI TaSI B&II Hind111 TuqI Hind111 MspI Hind111 MspI Haplo MspI Hind111 MspI TaqI EcoRI PVUII TaqI
ILZR DlOS31 DlOS33 DlOS32 DlOS35 DlOS17 DlOS39 DlOS28 VIM DlOS49 DlOS30 DlOS34 DlOS15 RBP3 DlOSll DlOS5 DlOS22 DlOS19 CDCP EGR2 DlOS16 DlOS45 DlOS14 DlOS29 DlOS13 DlOS62 DlOS4 DlOS20 DlOS18 DlOS12 DlOS37 DlOS43 OAT DlOS44 DlOS27 DlOS36 DlOS6 DlOS26 DlOS25
0.0 0.0 6.5 9.2 23.9 32.0 32.2 41.0 60.2 82.2 106.2 112.7 119.2 119.2 124.4 131.3 142.1 148.5 155.6 155.6 162.6 167.1 180.5 187.8 192.1 202.8 210.1 227.0 227.0 258.8 258.8 258.8 261.7 270.6 282.7 303.4 306.8 306.8 306.8
0.0 79.9* 89.4 101.9 115.7 122.4 122.4 127.5 133.5 136.9 144.3 147.8 147.8 150.4 150.4 155.2 157.1 163.4 167.7 167.9 173.4 173.4 182.7 185.6 188.2 188.2 200.0 208.5 222.6 229.4 229.4 229.4 234.0 234.0 241.3 253.7 274.1 277.5 278.1
r”“g
341 16 33 53 18 [8
*1 * 29 14 I31
301 [ * 451 4 17 2 40 5 6 1338 37
*I 36 44 15 [fi 22
211
of intragenic haplotype construction. [ 1, intergenic * Note. *, see text for definition whose order may be in doubt. ?, areas of low local support. ’ Local support = log,,, likelihood difference when adjacent pairs are reversed. * See discussion in text concluding this section.
often more precisely localize each of them with respect to the framework, we also performed the following analysis, using the “flips” option of CRI-MAP. Loci were first placed in their most likely interval in the framework, to obtain a candidate locus order incorporating all of the loci. Then, within this order, all permutations of each consecutive triplet of loci were constructed and the associated likelihood was computed. Table 3 lists the locus orders having relative odds within lOOO-fold of the best order.
haplotype-a
10 Data
set of contiguous,
Local support”
0.59 6.52 12.97 16.21 9.77 5.22 15.67 4.94 8.13 0.64 4.07 0.10 9.95 13.06 15.08 1.82
?
?
?
?
17.85 23.45 2.77 1.93 3.05 10.38 11.59 4.54 13.88
? ?
4.45 4.46 7.12 9.63 6.22
closely
linked
loci
Subsequent analysis revealed an anomaly concerning the support for placing DlOSll relative to RBPS. Although the “build” run described above uniquely placed DlOSll in the position shown in Fig. 3 at 1000 to 1 odds, this level of support appears to depend on the specific subset of loci used to perform the likelihood test. In particular, when DlOSll is instead tried in each possible position with respect to the entire set of chromosome 10 loci (using the order shown in Table 41, the odds favoring this placement of DlOSll drop
CEPH
CONSORTIUM
LINKAGE
to only 74 to 1, the next most likely position being on the other side of RBP3 between DlOS34 and DlOS30 (all other locations are still excluded at more than 1000: 1 odds). This appears to explain why four of the five groups analyzing the data did not uniquely place DlOSll with respect to RBP3. This anomaly may be a reflection of data errors, as several multiple crossovers in this region are revealed by the “chrompic” option of CRI-MAP. We have chosen to adopt (in Fig. 3) the unique location for DlOSll resulting from the “build” run in the expectation that random errors are unlikely to produce odds of 1000 to 1. The dataset used for this analysis contained the five corrected genotypes listed under Materials and Methods. No attempt was made to remove apparent RBP3 intralocus crossovers involving data for the probe cTBIRBP9. All likelihoods were calculated assuming no crossover interference and sex-equal recombination fractions. No information derived from physical localization studies was used in the map construction process. Chromosome 10 Map from the Galton Laboratory. Two maps were constructed, one with the program CRI-MAP (Donis-Keller et al., 1987) and one with MAP (Morton and Andrews, 1989). The maps were constructed independently, but with some comparison at various stages. The goal in each case was to produce as complete a map as the data permitted. Both approaches used the same genotypic data, which included haplotypes constructed by hand for RBPS, DlOSll, DlOS16, DlOS34, and DlOS43; to avoid apparent intragenic crossovers, all RBP3 data from individuals 140806 and 135007 and cTBIRBPS/TaqI data from family 1345 were omitted. EMBL3-93 (system 32) had been excluded from chromosome 10 by physical mapping at this laboratory and was not considered further. Original (uncorrected) genotypes were used for most of the construction process for both maps, and corrected data (see Materials and Methods) were incorporated only in the final stages. In preparation for CRI-MAP analysis, MAPMAKER (Lander et al., 1987) was used to provide pairwise sex-specific lod score tables and to suggest possible linkage groups. From the latter, a uniquely ordered triplet (DlOSlS-DlOSlS-D10S14) was identified by CRI-MAP and used as the basis for subsequent maps. The linkage groups and the physical map from HGMlO (Smith and Simpson, 1989) provided likely candidates that were tested, one at a time, with CRIMAP’s “all” option for incorporation into the map. A log,, likelihood support of at least 3.0 was required for the addition of a locus to the map. When all loci had been tested, those not fitted were tried again. DlOS27 and DlOS29 could be fitted only at a minimum log,, likelihood of 2.0, and three loci (pL28-5/XmnI,
MAP
OF
CHROMOSOME
10
405
H31/HindII, and M7/BamHI-M7/MspI, subsequently shown to be assigned to other chromosomes) could not be fitted at all. At various points in the analysis, two or more loci were grouped together as an intergenic haplotype, using CRI-MAP’s haplotype file facility. The criteria used were that the log,, likelihoods for adjacent positions of the test locus differed by less than 0.5, the distance between the loci was zero in one sex and less than 5 CM in the other, and the log,, likelihoods for the proposed haplotype were greater by at least 3.0 than those for alternate positions of the test locus. Six such haplotypes were eventually established and incorporated into the map. CRI-MAP allows recombination within a haplotype defined in this way, and the map distances in Table 4 reflect this. The remaining loci (ILBR, EGRB, and DlOSll) were then placed into the map in their most likely positions and the order was tested by permuting triplets down the length of the map. This produced two more likely orders, and only two other orders within a log,, likelihood difference of 3.0 from the best map. This best order, now based on the data containing the corrected genotypes, was tested, and the resulting, marginally better order was taken as final. Sex-specific map distances were calculated for this order, and local support was measured by inverting adjacent pairs of loci (Table 4). For the preparation of the second map, pairwise lod scores, factored by sex, were obtained from CRI-MAP and used as input to the MAP program. After global support for each locus was calculated (Morton and Andrews, 1989), three (pL28-5/XmnI, H31/HindIII, and M7/BamHI-M7/MspI) were excluded from further consideration since they yielded values less than 0.5, while the support for the remainder ranged between 14 and 340. An initial trial order was obtained from MAP (using a seriation algorithm), corrected on the basis of known physical evidence, and then MAP’s bootstrap facility was used to improve this order, with interference specified by a mapping parameter of 0.35 (Morton et al., 1985). Successive improvements were made by the bootstrap algorithm which, by permuting adjacent doublets in the map and adopting those resulting changes that exceed a given log,,, likelihood difference, seeks to maximize the overall likelihood, terminating when no further improvements can be made. The log,, likelihood difference was set low (O.l), since the intention was only to maximize likelihood. During the refinement process, megaloci were defined by combining pairs of contiguous, closely linked loci whose order could not be established. This was necessary to prevent the bootstrap algorithm from entering an infinite loop, and as MAP does not recognize any recombination within a megalocus, the distance between the component loci is 0 CM (Table 5). Locus
406
WHITE
ET
AL.
whose positions can be uniquely established with support of log,, likelihoods of 3.0 or more. The high degree of similarity between the two maps is encouraging, since it shows that the resulting order is largely independent of the strategy used to obtain it. Local support, calculated by entirely different methods, is not directly comparable, but differences between the two maps occur in areas of low local support in at least one map. Where differences in order exist, in the region between DlOS49 and DlOS5 and in the region between DlOS18
order of the map shown in Table 5 was constructed using the uncorrected data; using the corrected genotypes did not change the order. Only the distances and local support (derived from map distance and standard error; Morton and Andrews, 1989) were recalculated with data containing the corrected genotypes. The two maps represent complementary strategies: building by stepwise addition and attempting to refine a trial map toward the maximum likelihood order. Neither of the two maps presented here contains only loci
TABLE Chromosome
10 Map
from
the Galton
Laboratory:
5
MAP
(13) Analysis@
of CEPH Location
System 11
Probe
Enzyme
Locus
Female
Chromosome
10 Data
(CM) Local supportc
Male
ILZR cTBQ20 cTB14-16 cTBl4-15 pMHZ15 cTB14-36
BglI MspI RsaI TaqI MspI MspI
IL2R DlOS31 DlOS33 DlOS32 DlOS17 DlOS35
0.0 0.0 5.3 1.8 25.1 18.5
0.0 O.Ob 7.2 19.2 31.9 27.2
2.9 6.3 7.1 3.2
cmhlO-2 cTBQ7 hplf 1 CRI-J170 DlOS34 DlOSll pMCK2
B&II TaqI BclI PstI Haplo Haplo RsaI
DlOS39 DlOS28 VIM DlOS49 DlOS34 DlOS15 DlOSll
25.1 31.1 44.3 57.1 72.1 78.3 73.9
31.9 36.7 39.7 43.9 45.7 47.5 46.8
6.0 4.8 4.3 9.2 3.4 1.0
18 29 14
RBP3 cTBQ16 p9-12a pTBlO-163
Haplo MspI TaqI MspI
RBP3 DlOS30 DlOS5 DlOS22
78.3 83.1 87.1 94.1
47.5 48.5 51.9 52.4
3.3 3.0 2.3
r3: 301 * 45 4 17 2 40 5 6 3 [E
pTBlO-171 CDC2 zap32 DlOS16 CRI-J127 pTHH54 cTBQ-12 pTHHlOh1 CRI-JM14 pl-101 OS-2 pYNZ156 CRI-L368 HOATl-5
PUUII TQ41 Hind111 Haplo HindIII MspI WI BglII Hind111 Td Hind111 MspI Hind111 MspI
DlOS19 CDC2 EGR2 DlOS16 DlOS45 DlOS14 D10S29 DlOS13 DlOS62 DlOS4 DlOS20 DlOS18 DlOS12 OAT
105.5 100.9 105.5 109.3 116.5 125.1 129.8 132.9 139.0 146.1 155.7 158.0 181.5 180.1
59.7 56.8 59.7 64.3 64.3 68.5 70.2 72.4 72.4 82.3 87.8 93.4 106.5 102.7
3.7 1.7 5.3 2.0 0.9 1.0 4.8 4.6 0.5 7.9 2.1
37
H38 CRI-JlOl CRI-J125 cTBQ4 pMCT122-2
MspI Haplo Hind111 MspI TaqI
DlOS37 DlOS43 DlOS44 DlOS27 DlOS36
181.5 181.5 187.9 194.0 209.4
106.5 106.5 106.5 111.1 120.2
1.8 3.5 8.7
?
VTR4 pEFD75 pEFD70-2
EcoRI TaqI PVUII
DlOS6 DlOS25 DlOS26
211.6 211.6
137.9 142.5 142.5
5.7 1.3
?
191 25 24 r289
341 16 33 53 * L*s
*I
*1 44 15 20 I5241
221
of intragenic haplotype construction. [ ] megalocus, Note. *, see text for definition be in doubt. ?, areas of low local support. o Interference is allowed for using a mapping parameter p = 0.35. b See discussion in text concluding this section. ’ Local support based on map distance and standard error, (w/SE,)*/4.605 (11).
a set of contiguous,
closely
linked
?
2.1 5.6
loci whose
order
?
? ?
?
may
CEPH
and DlOS44,
CONSORTIUM
LINKAGE
at least one of the maps has low local support in the area, suggesting that the two orders are indistinguishable at the given level of local support. With respect to the two different orders in the former region, note the low level of local support for the position of DlOSll with respect to RBP3 on the map shown in Table 4 and with respect to DlOS34 on the second map (Table 5) and the low local support for DlOS34 with respect to DlOS30, also on the second map. The order of OAT and the megalocus containing DlOSl2 is inverted between the two maps, and there is evidence for low and marginal local support in their region on the second map (Table 5). The differences arise from the strategies used and, rather than indicating conflict, can be regarded as indicating regions of uncertainty in the map. Regions of maximum uncertainty are highly likely to be at the ends of the map, and the results given here by the two methodologies illustrate this problem. The difficulty in localization at the pter end of the maps presented here is compounded by a poorly informative terminal marker, ILBR. Differences between these complete maps, containing regions of order uncertainty, and the smaller consortium map may, in general, be explained by the nature of the data, as in some cases inserting one of a pair of loci will exclude the other and vice versa. The interval distances calculated by MAP are smaller than those obtained with CRI-MAP (Tables 4 and 5). Accordingly, the overall, multiple pairwise, male map length of 143 CM is closer to the 127 CM estimated from chiasmata counts in spermatogenesis (Morton et al., 1982) than that obtained by the multipoint analysis. Chromosome 10 Map from the Institute of Cancer Research. We have constructed a linkage map of the chromosome 10 markers typed in the consortium map database, principally using the computer program CRIMAP. CRI-MAP provides rapid computation of multipoint likelihoods, particularly for CEPH-type nuclear families, enabling large multipoint maps to be constructed (Donis-Keller et al., 1987). The 57 probe/enzyme combinations for which data were available included 43 distinct loci; corrected genotypes (see Materials and Methods) were used in constructing the map. Where more than one marker was available for the same locus, these were treated as haplotypes so that the markers were forced by the program to be placed consecutively, although not necessarily with zero recombination. In only one situation, however, was intralocus recombination observed; the cTBIRBP9 probe was recombinant with other markers at the RBP3 locus and is shown separately above RBP3 on the map. We first used the “build” option in CRI-MAP to search for the largest set of loci that could be uniquely
MAP
OF
CHROMOSOME
10
407
ordered with a likelihood ratio of at least 1OOO:l over other orders, adding loci successively to an initial linked pair of loci (DlOS33 and DlOS28). By this process 25 loci could be uniquely ordered-these correspond precisely to the loci ordered on the tentative consortium map before addition of DlOS15, DlOS17, and DlOS43. Note that in arriving at this ordered set of loci, the likelihoods calculated utilized ordering information from other markers not uniquely ordered, but placed in their various possible positions. This use of additional markers to provide indirect evidence of ordering allowed several more markers to be uniquely ordered than would otherwise have been possible. Likelihoods for possible placements of the remaining loci within this definitive order were then computed. The possible positions of each locus, with likelihoods of not smaller than l/lOOOth of the best order, are shown in Table 6. At this stage, 4 loci (H31, M7, EMBL3-93, and pL28-5) were discovered not to be placeable anywhere within the map, nor were they linked to any of the loci near the ends of the map. These loci were therefore discarded from the analysis, leaving 14 to be placed on the map. By reducing the tolerance of placement to lOO:l, and by checking likelihoods of various possible positions of loci in particular regions, it was possible to arrive at a maximum likelihood map for all 39 loci. As a check on this map, likelihoods were calculated for all orders obtained from this order by permuting adjacent triples of loci to search for orders with higher likelihood. In addition, for loci such as DlOS30 and DlOS27 which could be placed in two intervals that were not contiguous, complete maps were calculated under different assumed positions to check that the position with highest likelihood had been identified. Likelihoods for these final positions of the loci not definitely placed, compared to their next most favored placement, are shown in Table 6, and the resulting map is shown in Fig. 4. A male distance of 2.7 CM was found between DlOS39 and DlOS17 on this map. This distance, however, is uncertain, since DlOS39 is poorly informative. In fact, when the order of these two loci is inverted, the resultant map shows estimated sex-specific recombination frequencies of 0 for the interval and is only slightly less probable than the map presented here (odds of 1:1.3). Accordingly, we do not disagree with the placement of DlOS17 at the same position as that of DlOS39 on the consortium map. The genetic distances between adjacent loci on the map assume that recombination fraction and genetic distance are related via the Kosambi function. The total length of the map is estimated to be 275.8 CM in males and 309.8 CM in females. However, it is likely that these distances are overestimates for the following reasons: First, the dataset is likely to contain an unknown
408
WHITE
TABLE Possible
Positions
Locus
of Loci
Most
ET
AL.
6
Not Uniquely Ordered at 1OOO:l Odds on Chromosome from the Institute of Cancer Research
likely
interval
ILPR
[pter,
DlOS17 DlOS30
[DlOS39, [DlOS49,
DlOS28] DlOS34]
1.3:1 6.8~1
DlOS15 DlOSll EGRP
[RBP3, DlOS5] [RBP3, DlOS5] [DlOS22, DlOS19]
1:l 26:l 1.3:1
CDC2 DlOS45 DlOS13 DlOS37
[DlOS19, [DlOS19, [DlOS29, [DlOSlS,
DlOS16] DlOS16] DlOS62] DlOS12]
59:l 17:l 41:l 1:l
DlOS27 DlOS43 DlOS26 DlOS25
[DlOSlS, [DlOS12, [DlOS6, [DlOS6,
DlOS12] OAT] qter] qter]
17:l 1:l 2.0:1 2.0:1
D Ratio of likelihood obtained * Positions of the locus giving
by placing likelihoods
DlOS31]
Support”
locus in most likely interval, not smaller than l/lOOOth
number of erroneous typings, resulting in a spuriously high number of recombinant events. Interval distances between neighboring loci on the map are typically only a few centimorgans, so that most meioseswill not produce a recombination between a given locus and its nearest neighbor. Typing errors at a locus are therefore more likely to introduce a spurious recombinant than to eliminate a real one. Indeed, by using the “chrompic” option in CRI-MAP, a few individuals with implausibly large numbers of recombinant events could be identified. Second, the male map is inflated by the very large estimate for the distance between ILZR and DlOS31. This distance is extremely uncertain due to the poor informativeness of ILBR, and given the female recombination frequency of 0 it is reasonable to supposethat the true distance will be much smaller. Third, the estimated genetic distances on this map are much greater than would be suggestedby data on observed chiasmata distributions. Thus the distances for this map should be used only as a rough guide. Chromosome 10 Map from the Neurogenetics Laboratory, Massachusetts General Hospital. The corrected genotypic data for the 57 probe and enzyme systems were extracted from the CEPH chromosome 10 database into files conforming to the LINKAGE format. These files were converted into LIPED format files compatible with the LIPIN (Trofatter et aZ., 1986) data management system. Further conversion was necessary to create files acceptable to MAPMAKER (Lander et al., 1987). Data were checked at each stage of the conversion to ensure that no errors were introduced.
3.8:1
Other
10 Map
possible’
intervals
[DlOS31, DlOS33] [DlOS33, DlOS32] [DlOS35, DlOS39] [DlOS34, RBPB] [RBPB, DlOS5] [DlOS34, RBPB] [DlOS34, RBP3] [DlOS19, DlOSlS] [DlOS16, DlOS14] [DlOS22, DlOS19] [DlOS16, DlOS14] [DlOS14, DlOS29] [DlOS12, OAT] [OAT, DlOS44] [DlOS44, DlOS36] [DlOSlS, DlOS12] [DlOS36, DlOS6] [DlOS36, DlOS6]
divided by likelihood for the next best position. of the likelihood for the best position.
MAPMAKER was chosen as the analysis tool for two reasons. First, it can be faster than LINKAGE for calculations involving a large number of loci. Second, no other group participating in the consortium map was using MAPMAKER, thus allowing a diversity of analytic tools to be used on the same dataset. Our general approach for construction of the map proceeded as follows: First, all possible two-point lod scores were calculated from the genotypes for the 57 systems in the chromosome 10 database. (It was at this point that the loci defined by EMBL3-93, pL28-5, H31, and M7, later found to reside on other chromosomes, were discarded from further analysis, because they did not have substantially positive lod scores with any other markers in the database.) Superficial examination of two-point lod scores generally will reveal three to five very informative loci (determined by correspondingly high lod scores) residing in the samegeneral chromosomal region. All possible orders of these loci are tested, and a unique order (defined as a log,, likelihood difference of 3, odds of 1OOO:lor better, favoring this order) is defined. If no unique order is found, new starting loci are chosen. Additional loci are then tested in all possible positions against this defined map and are added if they have one position favored by odds of at least 1OOO:l. The resulting chromosome 10 map is presented in Fig. 5. Examination of the two-point lod scores allowed us to choose five loci (VIM, DlOS28, DlOS35, DlOS32, and DlOS33) that appeared to be spaced approximately 10 CM apart. All possible orders of these five loci were
CEPH
CONSORTIUM
LINKAGE
then tested. A log,, likelihood difference of 3.8 was found favoring a single order (Fig. 5). Additional markers were added to the map sequentially by testing them in every possible position against the established map. In all, 25 loci could be uniquely placed on this map. These loci define 21 unique positions, as DlOS12, DlOS37, and DlOS43 map to a single location, DlOS44 and OAT to another single location, and DlOS15 and RBP3 to still another single location. However, many loci could not be placed distinctly within this framework. Usually, these loci could be placed in one of two or three positions (Fig. 5) and could be significantly excluded from a large portion of the map. Because cTBIRBP9 demonstrated recombination with other RFLPs at the RBP3 locus, it was considered separately in determining locus order of the map. However, due to the small number of recombination events, it could not be placed in a unique position relative to the RBP3 locus (Fig. 5). Several points are worthy of consideration. First, MAPMAKER constructed a map similar to that generated by CRI-MAP and the LINKAGE package. However, MAPMAKER, as currently released, does not encourage users to examine sibships where either one or both parents are missing genotypic data, which occurred multiple times in this dataset. In fact, in the final analyses we were able to place some loci only after entering inferred parental data by hand. Unfortunately not all of the missing parental genotypes could be inferred, and thus only a subset (albeit a very large subset) of data could be analyzed. Some differences between our map and those of other laboratory groups are interesting. Two loci (DlOS14 and DlOS29) were placed with greater than 1OOO:l odds in a position inverted relative to the other maps. We are not entirely sure why this occurred, but it may indicate that the subset of data that MAPMAKER uses favors this alternative order, and thus may indicate internal inconsistencies within the entire dataset. Most other maps were able to uniquely localize several additional consortium map loci, including DlOS31, DlOS39, DlOS17, and DlOS34. These loci could not be placed on our map with the requisite 1OOO:l odds; in all cases they mapped to the positions given in the other maps with odds of at least 1OO:l. Our reduced level of support most likely arises from MAPMAKER not fully considering the overall dataset. Similarly, we observed no recombination between OAT and DlOS44 on multipoint analysis, probably a result of the data reduction. Second, the speed with which MAPMAKER can generate these maps can be truly impressive. On a VAX 8700, the 1596 two-point lod scores were calculated in less than 1 min, and the results easily displayed. Threeto five-point analyses generally took less than 3 min per order to calculate. However, several three- to five-
MAP
OF
CHROMOSOME
409
10
point analyses did take in excess of 30 min per order, depending on which markers were included in the analysis. This probably resulted from evaluation of phase-unknown intercross matings (both parents heterozygous for the same alleles) for the relevant loci. In all, the map was generated using approximately 4 days of CPU time on a VAX 8700. Finally, one concern that arises is that the mapping strategy we have employed, although generating an accurate map, does not necessarily generate the most inclusive map. An alternative set of initially mapped markers may allow a greater (or lesser) number of loci to be placed within a framework. This would depend on ambiguities of order (probably arising from an inevitable set of genotypic errors) being resolved by the removal of the offending locus. We have tested for a more complete set of mapped loci by trying different sets of starting markers and changing the order with which loci are entered into the map. This testing quickly became time-consuming and did not produce a more complete map. Comments
on Individual
Maps
This section summarizes various differences between the individual maps and the consortium map, and among the individual maps themselves, for locus order. In emphasizing differences here, it should be recognized that there is reasonable agreement among the individual maps and with the consortium map. The map from laboratory 1 (Fig. 2) contains DlOS27 and ILSR, whereas the consortium map does not. DlOS62 and DlOS6 on the consortium map are replaced by DlOS13 and DlOS25, respectively, on the map from this laboratory. These differences are explained by noting that DlOS27, DlOS25, DlOS13, and IL2R were not included in the subset of loci used to determine locus order on the consortium map; the first two loci were included on the initial map from laboratory 1. DlOS13 and DlOS25 are poorly localized on at least four individual maps (Figs. 3, 4, and 5 and Table 3), and DlOS27 is poorly localized on three maps (Figs. 3 and 4; see above, Chromosome 10 Map from the Galton Laboratory). ILBR, a poorly informative marker, was added to the final map from laboratory 1 because there was no recombination with DlOS31, the most pter marker on the initial map. ILBR is localized to the pter region of all of the other individual maps but its precise position on four maps is in doubt. Aside from the presence of DlOSll, the order of uniquely placed loci on the map from laboratory 2 (Fig. 3) is identical to that of the consortium map. The difficulty in placing this locus with respect to RBP3 on the consortium map is reflected in the inability to localize DlOSll with acceptable support on five individual maps, which may depend on which subset of loci
410
WHITE Female pter 77.6 9.5 12.5 13.9 4.7 2.7 4.5
ILiR I DlOS31 I D10S33 I DlOS32 I D10S35 I DlOS39 I DlOS17 I DlOS28
5.7
0.0 6.5 2.7 14.8 8.1 0.0 9.1 18.8
Viii
3.8 7.1 3.1 0.9 0.0 0.0 2.7 4.3 1.3 0.0
6.8
I
DlOS49 I DlOS30 I DlOS34
I
IRBP9 I RBP3 I DlOS15 I DlOSll I DlOS5 I D10S22 I EGR2
21.1 23.5 6.4 5.4 2.2 0.0 5.3 7.6 11.4 6.5
I
0.0
I
7.8
D10S19 4.0
CDC2 4.4
6.4 D10S45
2.2 9.4 3.0
6.8
I
DlOSl6 I DlOSl4
10.4
I
7.6
I
3.4
I
9.5
D10S29 2.1
DlOSl3 0.5 11.0 0.6 11.0 11.2 3.5 0.0 0.0 5.1
D10S62 I DlOS4
I
D10S20 I DlOS18 I DlOS27
I
DlOS37 I DlOS12 I DlOS43
I
16.8 0.0 0.0 3.9
15.5
I
2.2
I
0.0
I
0.0
DlOS6 DlOS26 0.6
32.8
I
D10S36 3.2
1.7
is used for its localization. For further discussion on this issue, seethe sections on the consortium map and the map from laboratory 2. Probably the most important difference found between the map from laboratory 5 and the consortium map is that of the order of two uniquely placed loci, DlOS29 and DlOSl4 (Fig. 5). The order on the former map is inverted with respect to that on the consortium map (and also on each of the other individual maps). This difference may result from the use of a (large) subset of genotypes from the CEPH families in the analysis by laboratory 5. As discussed in the section describing the map from this laboratory group, the program MAPMAKER usually omits data for analysis from families with one or both parents not genotyped. Another difference is the ambiguous localization of six consortium map loci on the individual map from this laboratory. Furthermore, DlOS37, DlOS27, and CDC2, which are not included on the consortium map, are uniquely placed on this individual map; in each case, at least three other laboratories were unable to incorporate these loci in their maps with acceptable support. These differences, too, may be attributable to the subset of genotypes used to construct the map from laboratory 5. It is difficult to compare the maps from laboratory 3 with the consortium map because, as noted above, different strategies were used to construct them. All 39 chromosome 10 loci were placed on the maps from laboratory 3 at their most likely positions, which were tested by measures of local support. Nonetheless, it is evident that the order of the 28 loci on the consortium map is identical to their order on these individual maps except for an inversion of (the megalocus containing) DlOS12/DlOS43 and OAT on the map prepared by multiple pairwise analysis. The group from laboratory 3 suggeststhat the observed difference reflects the uncertainty of the order in this region, as indicated by lbw levels of local support. None of the other maps from individual laboratories, including that from laboratory 3 prepared by CRI-MAP, show an order for DlOS12 and OAT different from that of the consortium map. Other possible evidence of uncertainty of order in this region is the ambiguous localization of DlOS37 (Figs. 2, 3, and 4) and of DlOS27 (Figs. 3 and 4).
10.1 DlilS44
21.2
16.8
AL.
OAT
0.0 13.5
8.7
ET
DlOS25
I
qter
FIG. 4. Chromosome 10 map from the Institute of Cancer Kesearch. The loci on this map are placed at their most likely positions, i.e., at positions giving the highest likelihoods. Loci shown in bold type could be uniquely ordered relative to one another, with odds of 1OoO:l or greater over other orders (see text). The estimated male and female genetic distances (in centimorgans) between adjacent pairs of loci are shown to the left and right of the figure. These distances are computed from recombination estimates by using the Kosambi mapping function.
CEPH Male
Female
CONSORTIUM
LINKAGE
MAP
OF
CHROMOSOME
411
10
pter I -----
.124
DlOS33
,036
.147
.151
.074
.116
.087
.197
1-
DlOS32
j-
DlOS35 DlOS28
.002
.247
.016
.176
.040
.112
.016
.llO
I-
I-
VIM
I-
DlOS49
) DlOS31 I
I 1 IL2R
; DlOSl7 I
RBP3/DlOS15
1DlOS30
IDlOS34
;DlOSll
I
I
1 I cTBIRBP9
DlOS5 I DlOS22 .027
.145
.119
,092
CDC2
1 EGRP DlOS16
.045
I
DlOSl9
.120
DlOS13
1DlOS45 I
DlOS29 .017
.046
.032
.190
.109
.045
. 100
.164
DlOS14 DlOS62 DlOS4 DlOS13 DlOS20 .136
.184
.073
.109
.054
.077
.004
.080
.305
.176
DlOS27 DlOS18 DlOS12/DlOS37/DlOS43 DlOS44/OAT
I -----
qter
DlOS6
i~l0~36
1DlOS25
I
I
I~l0~26
FIG. 5. Chromosome 10 map from the Neurogenetics Laboratory, Massachusetts General Hospital. The map represents all loci that could be placed with odds of 1OoO:l or better against every other position in the then current map. Loci to the right of the map have at least two positions, represented by the vertical lines, whose odds are within 1OOO:l of each other. All other positions for these loci are excluded with odds greater than 1OOOzl. The estimated male and female recombination fractions between adjacent pairs of loci, computed under the assumption of no interference, are shown to the left of the map. Loci that map to the same location (because no recombination was observed by two-point or multipoint analyses) are separated by a /.
The maps from the five contributing laboratories show differences in the region flanked by DlOS49 and DlOS5. All six individual maps agree with the order DlOS49, RBP3/DlOS15, and DlOS5 in this region. Additionally, DlOS34 was localized in the sameposition in this region on four maps (Figs. 2,3 and 4 and Table 5), giving an order agreeing with that of the consortium map (see Fig. 1 and Table 2). DlOSll was localized in this region, between RBP3/DlOS15 and DlOS5 (Fig. 3)) with acceptable support by a single laboratory. The two maps constructed by laboratory 3 placed DlOS30
within this region. However, the maps differed in the most likely position for this locus (Tables 4 and 5). Taken together, the maps from all the collaborating laboratories identify this region as one of uncertain order, especially with respect to the placement of DlOSll and DlOS30. Availability of CEPH Chromosome 10 Probe and Enzyme Genotypes With the publication of this CEPH consortium map, the genotypes in the chromosome 10 database are now
412
WHITE
available to the general scientific community. database may be obtained by contacting CEPH.
The
ACKNOWLEDGMENTS Author lists somehow do not succeed in completely conveying who contributed. In the case of the laboratories that collaborated in preparing the CEPH consortium map of chromosome 10, the difficulty in giving credit where it is due is compounded. Data collection and analyses would not have been possible without the scientific involvement and work of the following colleagues: at the Galton Laboratory and Imperial Cancer Research Fund Laboratory-Vince Andrews, John Attwood, Sue Povey, and Nigel Spurr; at the Institute of Cancer Research-C. Strong, B. Smith and K. Chin (for technical assistance), and B. Ponder (for support and encouragement); and at CEPH-we acknowledge the ongoing invaluable support and encouragement of Jean Dausset and Daniel Cohen, expert computer-generated artwork by Dustin Cartwright and Peter Cartwright, patient preparation of the manuscript by Elizabeth May, and dedication and commitment of the entire technical staff to supporting construction of the human genetic map. The following grant support is acknowledged: The Medical Research Council of Great Britain and the Cancer Research Campaign (to the Galton Laboratory and Institute of Cancer Research); NIH Grant NS20012 (to J.G., Neurogenetics Laboratory, Massachusetts General Hospital); NIH Grant l-ROI-GM40543 (to H.D.-K., Department of Genetics, Washington University School of Medicine); the Lucille P. Markey Charitable Trust (to P.G., Department of Genetics, Washington University School of Medicine); and Institut National de la Sante et de la Recherche Medicale, INSERM (to H.C. and M.L., CEPH). REFERENCES 1. BOWDEN, D. W., GRAVIUS, T. C., GREEN, P., FALLS, K., WURSTER-HILL, D., NOLL, W., MULLER-KAHLE, H., AND DONISKELLER, H. (1989). A genetic linkage mapof 32 loci on human chromosome 10. Gerwmics 5: 718-726. 2. DAUSSET, J., CANN, H., COHEN, D., LATHROP, M., LALOUEL, J-M., AND WHITE, R. (1990). Centre d’Etude du Polymorphisme Humain (CEPH): Collaborative genetic mapping of the human genome. Genomics 6: 575-577. 3. DONIS-KELLER, H., GREEN, P., HELMS, C., CARTINHOUR, S., WEIFFENBACH, B., STEPHENS, K., KEITH, T., BOWDEN, D., SMITH, D., LANDER, E., BOTSTEIN, D., AKOTS, G., REDIKER, K., G~~vrus, T., BROWN, V., RISING, M., PARKER, C., POWERS, J., WA?T, D., KAUFFMANN, E., BRICKER, A., PHIPPS, P., MULLER-KAHLE, H., FULTON, T., NG, S., SCHUMM, J., BRAMAN, J., KNOWLMN, R., BARKER, D., CROOKS,S., LINCOLN, S., DALY, M., AND ABRAHAMSON, J. (1987). A genetic linkage map of the human genome. Cell 51: 319-337. 4. FARRER, L. A., CASTIGLIONE, C. M., KIDD, J. R., MYERS, S., CARSON,N., SIMPSON, N. E., AND KIDD, K. K. (1988). A linkage group of five DNA markers on human chromosome 10. Genomics 3: 72-77. 5. LANDER, E. S., GREEN, P., ABRAHAMSON, J., BARLOW, A., DALY, M. J., LINCOLN, S. E., AM) NEWBURG, L. (1987). MAPMAKER: An interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1: 174-181. 6. LATHROP, G. M., LALOUEL, J-M., JULIER, C., AND On, J. (1985). Multilocus linkage analysis in humans: Detection of linkage and estimation of recombination. Amer. J. Hum. Genet. 37: 482-498.
7. LATHROP, G. M., NAKAMURA, Y., CARTWRIGHT, P., O’CONNELL, P., LEPPERT, M., JONES, C., TATEISHI, H., BRAGG, T., LAU)UEL,
ET
AL.
J-M., AND WHITE, R. (1988). A primary genetic map of markers for human chromosome 10. Genomics 2: 157-164. 8. MATHEW, C., CHIN, K., EASTON, D., THORPE, K., CARTER, C., LIOU, G., FONG, S-L., BRIDGES, C., HAAK, H., KRUSEMAN, A., SCHI~R, S., HANSEN, H., TELENIUS, H., TELENIUS-BERG, M., AND PONDER, B. (1987). A linked genetic marker for multiple endocrine neoplasia type 2A on chromosome 10. Nature (London) 328: 527-528. 9. MORTON, N. E., LINDSTEN, J., ISELIUS, L., AND YEE, S. (1982). Data and theory for a revised chiasma map of man. Hum. Genet. 62: 266-270. 10. MORTON, N. E., MACLEAN, C. J., AND LEW, R. (1985). Tests of hypotheses on recombination frequencies. Genet. Res. 45: 279-286.
11. MORTON, N. E., AND ANDREWS, V. (1989). MAP, an expert system for multiple pairwise linkage analysis. Ann. Hum. Genet. 53: 263-269. 12. NAKAMURA, Y., LATHROP, M., BRAGG, T., LEPPERT, M., O’CONNELL, P., JONES, C., LALOUEL, J-M., AND WHITE, R. (19888). An extended genetic linkage map of markers for chromosome 10. Genomics 3: 389-392. 13. NAKAMURA, Y., LATHROP, M., O’CONNELL, P., LEPPERT, M., BARKER, D., WRIGHT, E., SKOLNICK, M., KONDOLEON, S., Lrrr, M., LALOUEL, J-M., AND WHITE, R. (1988b). A mapped set of DNA markers for human chromosome 17. Genomics 2: 302309. 14. NAKAMURA, Y., LATHROP, M., O’CONNELL, P., LEPPERT, M., LALOUEL, J-M., AND WHITE, R. (1988c). A primary mapof ten DNA markers and two serological markers for human chromosome 19. Gerwmics 3: 67-71. 15. NAKAMURA, Y., MATHEW, C. G. P., SOBOL, H., EASTON, D. F., TELENIUS, H., BRAGG, T., CHIN, K., CLARK, J., JONES, C., LENOIR, G. M., WHITE, R., AND PONDER, B. A. J. (1989). Linked markers flanking the gene for multiple endocrine neoplasia type 2A. Genomics 6: 199-203. 16. O’CONNELL, P., LATHROP, G. M., LAW, M., LEPPERT, M., NAKAMURA, Y., HOFF, M., KUMLIN, E., THOMAS, W., ELSNER, T., BALLARD, L., GOODMAN, P., AZEN, E., SADLER, J. E., CAI, G. Y., LALOUEL, J-M., AND WHITE, R. (1987). A primary genetic linkage map for human chromosome 12. Genomics 1: 93-102. 17. Paris Conference (1971). “Standardization in Human Cytogenetics. Birth Defects: Original Article Series,” Vol. 8, p. 7, The National Foundation, New York. 18. REEDERS, S., KEITH, T., GREEN, P., GERMINO, G., BARTON, N., LEHMANN, O., BROWN, V., PHIPPS, P., MORGAN, J., BEAR, J., AND PARFREY, P. (1988). Regional localisation of the autosomal dominant polycystic kidney disease locus. Genomics 3: 150-155. 19. SIMPSON, N., KIDD, K., GOODFELLOW, P., MCDERMID, H., MYERS, S., KIDD, J., JACKSON, C., DUNCAN, A., FARRER, L., BRASCH, K., CASTIGLIONE, GREENBERG, C., GUSELLA,
C., GENEL, J., HOLDEN,
M., GERTNER, J., AND WHITE,
J., B.
(1987). Assignment of multiple endocrine neoplasia type 2A to chromosome 10 by linkage. Nature (London) 328: 528-530. 20. SMITH, M., AND SIMPSON, N. E. (1989). Report of the committee on the genetic constitution of chromosomes 9 and 10, Human Gene Mapping 10 (1989), Tenth International Workshop on Human Gene Mapping. Cytogenet. Cell Genet. 61: 202-225. 21. TROFA?TER, J. A., GAINES, J. L., AND CONNEALLY, P. M. (1986). LIPIN: An interactive data entry and management program for LIPED. Am. J. Hum. Genet. 39: 147-148. 22. WARNICH, L., DIETZSCH, E., HERBERT, J., BRUSNICKY, J., LAUBSCHER, L., AND RETIEF, A. (1989). An anonymous DNA probe (H38) [DlOS37] detects a MspI polymorphism on chromosome 10. Nucleic Acids Res. 17: 474.
