GENOMICS
8,
l-6 (1990)
A Genetic JONATHAN
Linkage Map of Chromosome
L. HAINES,* LAURIE J. OZELIUS,* HEATHER MCFARLANE,* FRANK MARTINIuK,t
*Neurogenetics
Laboratory, toepartment
ROCHELLE tiIRSCHHORN,t
Received
September
1, 1989;
revised
ANIL MENON,*
AND JAMES
Massachusetts General Hospital, and Harvard Medical of Medicine, New York University School of Medicine, March
17
F.
STEPHANIE TzALL,t
GUSELLA*
School, Boston, Massachusetts New York, New York 10003
02114; and
30, 1990
relatively high-resolution maps of small regions. We initially developed the current map to help localize the NFl gene, and fortuitously, it may now also be used for the localization of the CMT2 gene. This map will also be useful for the examination of translocations and other chromosomal rearrangements and will provide a set of mapped markers available for use in mapping currently unlocalized disease genes. Finally, because our map is based on an almost completely independent data set, it can be combined with data from previous maps to provide a more accurate, more densely populated map.
We have developed a genetic linkage map of 19 markers (including nine genes) on human chromosome 17, providing 13 reference points along virtually the entire length of this chromosome. The map covers an estimated 149 CM in length (sex-averaged), with a total length of 214 CM in females and 96 CM in males. This sex difference appears to be significant along virtually the entire length of the map. This map will be useful both for providing reference points for fine structure genetic and physical mapping and for genetic linkage studies of diseases, including von Recklinghausen neurofibromatosis and Charcot-Marie-Tooth disease. a leso Academic press, IW.
MATERIALS
AND METHODS
Venezuelan Reference Pedigree
INTRODUCTION
We have identified many large sibships useful for genetic linkage reference mapping. These have been collected as part of our ongoing studies of Huntington disease in a very large Venezuelan kindred (Tanzi et al., 1988). Data for all markers were generated on 18 closely related sibships as previously described (Tanzi et al., 1988). Two of these sibships are also included in the CEPH collaboration (CEPH codes 102 and 104). We have recently expanded our reference set of pedigrees to include 73 sibships now comprising over 1100 potentially informative meioses. There is no overlap with the CEPH in these newer sibships. A subset of markers (D17S71, D17S57, D17S73, D17S33, and D17S58) spanning the region that includes the NFl gene was typed on most of the expanded reference set.
In 1980 it was proposed (Botstein et al.) that restriction fragment length polymorphisms (RFLPs) could be used as reference markers for the generation of genetic linkage maps of entire chromosomes. Since that time, a large number of genetic linkage maps have been published (e.g., Drayna and White, 1985; Donis-Keller et al., 1987; Tanzi et al., 1988). These maps serve a dual purpose. First, they serve as a reference source for determining the best set of markers available for identifying the chromosomal location of human disease genes.Second, these maps can serve as reference points for generating physical maps and comparing genetic and physical distances. Chromosome 17 has engendered considerable interest since the mapping of von Recklinghausen neurofibromatosis (NFl) (Barker et al., 1987; Seizinger et al., 1987) and one form of Charcot-Marie-Tooth disease(CMT2) (Vance et al., 1989) to the pericentromeric region and the identification of the TP53 gene as the probable site of mutations predisposing toward colorectal cancer (Vogelstein et al., 1989; Baker et al., 1989). Several other chromosome 17 maps have been published (Ruano et al., 1988; Nakamura et al., 1988; Goldgar et al., 1989), providing either widely spaced reference points along large portions of the chromosome or
Probes Methods for probe preparation, DNA digestion, Southern blotting, and hybridization have been described in detail elsewhere (Tanzi et al., 1988). Table 1 provides detailed descriptions of each probe. Genetic Linkage Analysis All data were entered into a computer file using the LIPIN (Trofatter et al., 1986) data management pro1 Copyright 0 1990 All rights of reproduction
osss-7543/90 $3.00 by Academic Press, Inc. in any form reserved.
2
HAINES
ET
TABLE Information Prohe pYNZ22.1 pHF12-2
Designation D17S5 D17Sl
p53-H7
TP53
pHRpII5.5 pA1041
POLRP D17S71
LEW301 pHHH202 pEW207
D17S58 D17S33 D17573
LEW206 p9TAU LEW203 HPP pE51
D17S57 MTBTl D17S54 PPY NGFR
pCMM86 chaH800
D17S74 GH
pHtk9
TKl
GAA67 BS3 pTHH59
GAA HOX2 D17S4
on Markers
Enzyme MspI &XI
MspI Haplotype SCUI B&II Haplotype Hind111 MspI PUUII Haplotype Tag RsaI B&I Hind111 Huplotype MspI XbaI B&I MspI HincII XmnI XmnI MspI HincII B&II BglII Hind111 Sea1 BstEII Haplotype Sad Taq
gram. Data can then be modified and converted into LIPED, LINKAGE, and MAPMAKER formats with ease.The sibships were entered as nuclear families (including, where possible, grandparents). This allows the use of more efficient algorithms for analysis and results in little loss of linkage information (Haines, unpublished results). All linkage analyses were performed using the MAPMAKER program (version 1.0) (Lander et al., 1987). Our general procedure is to generate all possible two-point lod scores and examine them to determine a set of three to four markers that appear to be well spaced (5-15 CM apart) and mutually informative. We then examine all possible orders to determine the most likely order, and accept that as definitive if it has odds greater than 1OO:l better than any other order. New markers are then tried in every possible position along the current map and are placed only if the odds of one position are at least 1OO:l better than those of any other position on the map. In this way, we can build a uniquely ordered set of markers. All order determinations were performed using equal recombination fractions in both sexes.
AL.
1
Used for Chromosome Allele
frequency
VNTR 0.69 0.31 0.65 0.35
17 Observed
heterozygosity
Ref.
0.86
(21) (24
0.63
(16) 0.77 0.06 0.17 0.00 0.22 0.78 0.50 0.50 0.19 0.81 0.00 0.61 0.08 0.31 0.40 0.60 0.57 0.43 0.80 0.20 0.75 0.25 0.33 0.45 0.22 0.00 0.30 0.70 0.78 0.22 0.66 0.34 0.52 0.48 0.64 0.34 0.92 0.08 0.02 0.98 VNTR 0.67 0.33 0.25 0.75
0.54 0.46
(31)
(23) 0.50 0.43 0.35
0.60 0.36 0.29 0.42 0.42 0.33
0.90 0.50
(2) 03) (10)
(2) (2’0 (2) (11)
(6) (22) (7)
U&20) 0.34 0.02 0.64 0.03 0.05 0.24 0.68 0.85 0.15 VNTR
0.71 0.42 0.29 0.76
(15) (1%
(21)
Early in this process, we realized that errors in the data could have a profound effect on the ability to place a marker uniquely, as those errors would most likely be detected as double recombination events. To facilitate error checking, we have developed the program CHROMLOOK (Haines, in preparation), which examines the inheritance of each marker (given a particular marker order) and determines the probable maternal or paternal origin of each child’s alleles. The output consists of paternal and maternal alleles for each child, with recombination events marked. Almost all recombination events can be detected in this way. Apparent occurrences of double recombinations were checked against the original autorads for errors, and many such events were removed by entering the corrected data. In a few cases,these events were resolved only when markers were retyped. This extensive error checking allowed us to increase the number of markers with unique positioning from 10 to 13. Sex-specific lod scoreswere calculated after the final map was determined. Differences were tested for significance using the likelihood ratio test. The -2 In likelihood approximates a X2 distribution where the
LINKAGE
MAP
OF
number of degrees of freedom is determined by the difference in the number of parameters estimated. RESULTS
Table 2 presents the peak recombination fractions and lod scores for all possible two-point combinations. These data were used to identify a set of four markers (NGFR, GH, TKl, and GAA) that produced a unique ordering and was the starting set on which the rest of the map was built. The choice of this set of markers was somewhat arbitrary; a similar set of markers elsewhere on the chromosome could have been used instead. Figure 1 presents the sex-averaged and sex-specific maps, along with physical localizations of some of the markers. Three markers could not be placed uniquely within the final map. D17S4 may reside on either side of TKl; its preferred position is between GH and TKl, but the odds are only l&l over a position between TKl and GAA. D17S57 could be on either side of D17S73; the odds are essentially equal for either position. HOX2 could reside in any position from D17S73 distal to D17S74. Its preferred position is between D17S54 and PPY, with odds over the next best position being 16: 1. Since these markers were not uniquely placed, they were not used in the tests of recombinational sex differences. Three loci in the map, TP53, POLRB, and D17S1, are very close to one another, and their relative order could not be determined. In fact, only the orders D17S5-D17Sl-TP53-POLR2-D17S71 and D17S5TP53-D17Sl-POLR2-D17S71 could be rejected at 1OO:l odds (Table 3). Of the 22 triply informative
TABLE Two-Point Marker
S5
55 TP53 POLR2 Sl s71 558 533 573 MTBTl s54 PPY NGFR s74 GH TKl GAA s57 54 HOX2
3.04 5.00 3.92 0.00 0.85 0.12 0.13 0.00 0.03 0.21 0.00 0.47 0.12 0.16 0.13 0.10 0.00 0.00
a Not
TP53 0.28
informative.
16.39 9.05 1.35 1.80 0.14 0.74 1.26 0.76 0.86 2.57 1.96 0.13 0.00 0.14 0.65 0.00 0.03
POLR2 0.24 0.01 15.07 3.66 1.30 0.18 0.13 1.19 0.53 0.09 2.23 1.48 2.82 0.99 0.00 0.48 0.11 0.60
Lod Scores Sl 0.28 0.05 0.03 1.68 0.68 0.00 0.24 0.66 2.20 0.15 2.62 0.62 0.52 0.00 0.00 0.55 0.00 0.00
(Below
Diagonal)
571
S58
s33
573
0.50 0.33 0.24 0.28
0.33 0.20 0.23 0.29 0.08
0.44 0.34 0.39 0.50 0.11 0.03
0.45 0.36 0.42 0.35 0.11 0.05 0.06
21.96 13.65 27.50 0.00 18.37 0.00 3.93 2.68 1.47 0.65 0.00 24.40 0.00 1.02
22.04 25.81 2.84 15.50 3.74 4.63 8.49 3.02 1.31 0.18 12.06 0.39 1.13
19.60 3.24 18.35 0.09 2.58 1.36 0.22 1.85 0.36 17.69 2.58 0.95
3.90 34.43 2.54 3.65 4.53 0.98 0.89 1.01 41.20 0.11 0.51
MTBTl 0.50 0.26 0.16 0.32 0.50 0.14 0.12 0.10
CHROMOSOME
3
17
events, only two crosses were observed, both arguing for the order D17S5-TP53-POLR2-D17Sl-D17S71. However, all three loci are included in the map because each is necessary to obtain more accurate map distances given the distribution of informativeness of these markers relative to the flanking markers D17S5 and D17S71. The overall female to male ratio of recombination fraction along the entire map is 2.25:1. We tested for significance of the sex differences using two different models. If we let each interval vary independently, the ~‘(15) value is 71.42 (P < 0.005), indicating that there is significant variation by sex compared to a null hypothesis of a 1:l sex ratio across the entire map. We then examined the possibility that only one or two regions might be responsible for this difference. We examined the data by defining three regions: D17S5D17S58, D17S58-NGFR, and NGFR-GAA. In each case [X2(5) = 27.4, P < 0.005; x’(6) = 23.67, P < 0.005; x2(4) = 20.35, P < 0.005, respectively] the sex differences are statistically significant compared to a 1:l sex ratio. With one nonsignificant exception (TKl-GAA) all intervals had larger female than male recombination fractions. DISCUSSION
We have generated a linkage map with 13 distinct reference points extending virtually the entire length of chromosome 17. Four of the nine genes we have mapped (TP53, POLRB, MTBTl, and GAA) have not, to our knowledge, been previously placed on a genetic linkage map. The remaining five genes (NGFR, TKl,
2 and Peak S54 0.44 0.32 0.34 0.25 0.11 0.17 0.06 0.02 0.00
PPY 0.42 0.24 0.32 0.34 0.50 0.14 0.42 0.18 0.10 0.19
0 Values NGFR 0.50 0.20 0.20 0.23 0.16 0.10 0.18 0.05 0.09 0.03 0.05
(Above
Diagonal)
574
GH
0.41 0.30 0.32 0.36 0.29 0.17 0.32 0.22 0.11 0.16 0.24 0.12
0.45 0.40 0.24 0.36 0.32 0.20 0.36 0.29 0.17 0.24 0.45 0.20 0.09
TKl 0.45 0.50 0.33 0.50 0.38 0.32 0.28 0.33 0.31 0.31 0.47 0.39 0.29 0.23
GAA
S57
S4
0.43 0.34 0.50 0.50 0.50 0.38 0.36 0.27 0.30 0.40 0.27 0.35 0.29 0.26 0.11
0.42 0.50 0.32 0.33 0.08 0.05 0.03 0.00 0.00 0.02 0.24 0.13 0.24 0.35 0.50 0.50
0.50 0.50 0.43 0.50 0.50 0.38 0.25 0.45 0.25 0.33 0.44 0.27 0.31 0.22 0.01 0.12 0.50
8.43 3.59 7.04 8.96 4.23 0.92 0.49 0.90 2.86
1.32 8.81 7.45 2.07 1.18 0.14 32.96 1.23
5.53 3.18 0.03 0.02 0.50 0.88 0.05
9.57 5.15 0.15 0.07 3.81 2.78
21.53 4.04 1.16 2.98 3.45
5.60 1.35 0.23 6.36
9.06 0.00 36.25
0.00 7.25
0.00
0.30
0.75
0.39
1.20
2.24
0.97
1.00
0.04
0.54
a
HOX2 0.50 0.31 0.00 ‘0.50 0.13 0.07 0.12 0.13 0.00 0.18 0.10 0.00 0.14 0.17 0.17 0.32 0.20, (I
HAINES
/
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ET
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AL.
31
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/// $5 /
'//
'5
'//
'//
46
--N
\
\
' \
\ \
\ \
D17S73 MTBTll 017854
--_ II
-_ --
\
Dl7S50 D17S33
IO
---_ ------>
25
D17S71
- - 3'
_--___---23 24
F
, /. / , / / 17
/
22
D17S5
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--_ - .
HOX2
NGFR
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D17S74 17
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GH
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32
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D17S4 TKl
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FIG. mapping mapping
Dl7S57
PPY
,5
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1
d
t
GAA
?
1. Genetic linkage map of chromosome 17 showing male, sex-averaged, and female maps. Distances are indicated in CM (Kosambi function). Physical mapping of some markers is indicated by the square brackets on the idiogram. Markers with only regional linkage are indicated on the far right; the tick marks show the most preferred positions.
HOX2, PPY, and GH) have never appeared in the same genetic linkage map before. Our map, based on a separate data set, has confirmed the order and, more roughly, the distance estimates of maps based on the CEPH or other pedigrees. Tsipouras et al. (1988) attempted to map GH and COLlAl relative to the centromere, but were unable to generate a unique statistically significant order. Two preliminary reports (Miki et al., 1987; Ruano et al., 1988) have presented data for linkage of HOX2, NGFR, and PPY. Our results are entirely consistent with
theirs. In fact, the distance between PPY and NGFR in both reports (5 CM) is very close to our own estimate of 4 CM. Our inability to localize HOX2 results from its relative lack of heterozygosity in the Venezuelan Reference Pedigree. Ruano et al. (1988) reported no recombination between HOX2 and NGFR in approximately 45 informative events. We also observe no recombination, but have only four mutually informative events. HOX2 was placed proximal to NGFR with odds of 27:l over a distal position by Goldgar et al. (1989). Their 6 estimates indicate that recombination between
LINKAGE
TABLE Orders
and Relative D17S1, Order
D17S5-TP53-POLR2-D17Sl-D17S71 D17S5-D17Sl-POLR2-TP53-D17S71 D17S5-POLR2-TP53-D17Sl-Dl7S71 D17S5-POLR2-D17Sl-TP53-D17S71 D17S5-D17Sl-TP53-POLR2-D17S71 D17S5-TP53-D17Sl-POLR2-D17S71
MAP
OF
3
Likelihoods and POLRZ
for TP53,
Relative log,, (likelihood) 0.00 -0.57 -1.21 -1.28 -2.31
-2.40
HOX2 and NGFR has been observed. This is interesting in light of the report of Bentley et al. (1989) which places HOX2 and NGFR on the same 590-kb fragment. This may indicate an increased rate of recombination in this region, at least in families segregating NFl. The map of Donis-Keller et al. (1987) includes only one marker used in the current map, thus making comparisons between the two impossible. Recently another linkage map of chromosome 17 was published (Nakamura et al., 1988). The current map shares five markers in common, and these are spread across virtually the entire length of the map, facilitating comparison. Not surprisingly, order is consistent between these maps. However, there is some variation in genetic distance as estimated from the two data sets. In particular, the distance between D17S33 and D17S74 (34 CM vs 44 CM) and the distance between D17S74 and D17S4 (34 CM vs 56 CM) are smaller in our data set. The possible reasons for these differences are many and include true underlying variation and differences in methods of manipulating and analyzing the data. However, the most likely explanation is simply that these differences arise from random sampling variation in the selection of the families being studied. Both maps show a significant excess of female recombination throughout the majority of the map, although this is more pronounced in our data set (female/male ratio 2.251 vs 1.54:l). The excess male recombination observed by Nakamura et al. (1988) was observed only at the telomeres, in regions not covered by the current map. The pericentromeric region of this chromosome has received a tremendous amount of attention since the recent mapping of NFl to 17q11.2. Goldgar et al. (1989) have produced a summary map of this region based on data from families segregating NFL The differences between the current map and that one are small and exist only in the estimates of 13.It is interesting to note that Goldgar et ab (1989) observed a ~-CM distance between D17S57 and D17S73, while we have observed only one recombination event in approximately 200 mutually informative meioses. One must be aware of the possibility that the presence of disease mutations
CHROMOSOME
5
17
in the region being studied may have some impact on the resulting recombination fraction estimates. Thus, it is unclear whether this observed difference is the result of random chance or is of underlying biological significance. This map will be useful for refining the genetic position of the NFl gene and for providing distances necessary for using markers in prenatal diagnosis of this disease. Similarly, refining the genetic localization of CMT2 in the pericentromeric region of this chromosome may also make use of these data. In particular, by using an essentially independent data set for generation of our map, it is now possible to combine our data with those of other groups to produce a denser, more refined, and more accurate genetic map of chromosome 17. This refined map will serve as a useful first step toward the eventual goal of providing a complete physical map of this chromosome and, ultimately, toward the eventual sequencing of the human genome. ACKNOWLEDGMENTS We gratefully thank Drs. Y. Nakamura, D. Barker, P. Murphy, T. Takeuchi, F. Ruddle, P.-F. Lin, R. White, D. Wolf, and R. Neve for kind gifts of their probes. S.T. is a postdoctoral fellow of the Arthritis Foundation. This work was supported in part by NIH Grants NS20012 and NS22031.
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ET
AL.
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P., LEPPERT, M., BARKER, D., LATHROP, M., CARTWRIGHT, P., R. (1987). A primary genetic map Cell Genet. 46: 668.
22. NAKAMURA,
Y., MARTIN, C., MYERS, R., BALLARD, L., LEPPERT, M., O’CONNELL, P., LATHROP, G. M., LALOUEL, J.-M., AND WHITE, R. (1988). Isolation and mapping of a polymorphic DNA sequence (pCMM86) on chromosome 17q (D17S74). Nucleic Acids Res. 16: 5223.
23. NAKAMURA,
Y., LATHROP, M., O’CONNELL, P., LEPPERT, M., BARKER, D., WRIGHT, E., SKOLNICK, M., KONDOLEON, S., LI?T, M., LALOUEL, J.-M., AND WHITE, R. (1988). A mapped set of DNA markers for human chromosome 17. Genomics 2: 302309.
24. NAYLOR,
S. L., SAKAGUCHI, A., BARKER, D., WHITE, R., AND SHOWS, T. B. (1984). DNA polymorphic loci mapped to human chromosomes 3, 5,9, 11,17, 18, 22. Proc. Natl. Acad. Sci. USA
81:2447-2451. 25. RUANO, G., MIKI,
T., FERGUSON-SMITH, A., RUDDLE, F. H., AND KIDD, K. K. (1988). Tight linkage of HOX2 and NGFR and linkages with five other gene on 17q. Amer. J. Hum. Genet. 43: A157.
26. SEIZINGER,
B. R., ROULEAU, G. A., OZELIUS, L. J., LANE, A. H., FARYNIARZ, A. G., CHAO, M. V., HUSON, S., KORF, B. R., PARRY, D. M., PERICAK-VANCE, M. A., COLLINS, F. S., HOBBS, W. J., FALCONE, B. G., IANNAZZI, J. A., ROY, J. C., ST. GEORGE-HYSLOP, P. H., TANZI, R. E., BOTHWELL, M. A., UPADHYAYA, M., HARPER, P., GOLDSTEIN, A. E., HOOVER, D. L., BADER, J. L., SPENCE, M. A., MULVIHILL, J. J., AYLSWORTH, A. S., VANCE, J. M., ROSSENWASSER, G. 0. D., GASKELL, P. C., ROSES, A. D., MARTUZA, R. L., BFZEAKEFIELD, X. O., AND GUSELLA, J. F. (1987). Genetic linkage of von Recklinghausen neurofibromatosis to the nerve growth factor receptor gene. Cell 49: 589594.
27. TANZI, R. E., BIRD, E. D., LATT, S. A., AND NEVE, R. L. (1987). The amyloid beta protein patients with Alzheimer’s
gene is not duplicated in brains disease. Science 238: 666-669.
from
28. TANZI, R. E., HAINES,
J. L., WATKINS, P. C., STEWART, G. D., WALLACE, M. R., HALLEWELL, R., WONG, C., WEXLER, N. S., CONNEALLY, P. M., AND GUSELLA, J. F. (1988). Genetic linkage map of human chromosome 21. Genomics 3: 129-136.
29. TROFATTER,
J. A., HAINES, J. L., AND CONNEALLY, P. M. (1986). LIPIN: An interactive data entry and management program for LIPED. Amer. J. Hum. Genet. 39: 147-148.
30. TSIPOURAS, H. F. (1988). arm of human 110.
P., SCHWARZ, R. C., PHILLIPS, J. A., AND WILLARD, A centromere-based linkage group on the long chromosome 17. Cytogenet. Cell Genet. 47: lOS-
31. VANCE, J. M., NICHOLSON,
G. A., YAMAOKA, L. H., STAJICH, J., STEWART, C. S., SPEER, M. C., HUNG, W. Y., ROSES, A. D., BARKER, D., AND PERICAK-VANCE, M. A. (1989). Linkage of Charcot-Marie-Tooth neuropathy type la to chromosome 17. Exp. Neural. 104: 186-189.
32. VANTUINEN,
P., AND LEDBETTER, D. H. (1987). Construction and utilisation of a detailed somatic cell hybrid mapping panel for human chromosome 17: Localization of an anonymous clone to the critical region of Miller-Dieker syndrome, deletion 17~13. Cytogenet. Cell Genet. 46: 708-709.
33. VOGELSTEIN,
B., FEARON, E. R., KERN, S. E., HAMILTON, S. R., PREISINGER, A. C., N~RA, Y., AND WHITE, R. (1989). Allelotype of colorectal carcinoma. Science 244: 207-211.
8,
l-6 (1990)
A Genetic JONATHAN
Linkage Map of Chromosome
L. HAINES,* LAURIE J. OZELIUS,* HEATHER MCFARLANE,* FRANK MARTINIuK,t
*Neurogenetics
Laboratory, toepartment
ROCHELLE tiIRSCHHORN,t
Received
September
1, 1989;
revised
ANIL MENON,*
AND JAMES
Massachusetts General Hospital, and Harvard Medical of Medicine, New York University School of Medicine, March
17
F.
STEPHANIE TzALL,t
GUSELLA*
School, Boston, Massachusetts New York, New York 10003
02114; and
30, 1990
relatively high-resolution maps of small regions. We initially developed the current map to help localize the NFl gene, and fortuitously, it may now also be used for the localization of the CMT2 gene. This map will also be useful for the examination of translocations and other chromosomal rearrangements and will provide a set of mapped markers available for use in mapping currently unlocalized disease genes. Finally, because our map is based on an almost completely independent data set, it can be combined with data from previous maps to provide a more accurate, more densely populated map.
We have developed a genetic linkage map of 19 markers (including nine genes) on human chromosome 17, providing 13 reference points along virtually the entire length of this chromosome. The map covers an estimated 149 CM in length (sex-averaged), with a total length of 214 CM in females and 96 CM in males. This sex difference appears to be significant along virtually the entire length of the map. This map will be useful both for providing reference points for fine structure genetic and physical mapping and for genetic linkage studies of diseases, including von Recklinghausen neurofibromatosis and Charcot-Marie-Tooth disease. a leso Academic press, IW.
MATERIALS
AND METHODS
Venezuelan Reference Pedigree
INTRODUCTION
We have identified many large sibships useful for genetic linkage reference mapping. These have been collected as part of our ongoing studies of Huntington disease in a very large Venezuelan kindred (Tanzi et al., 1988). Data for all markers were generated on 18 closely related sibships as previously described (Tanzi et al., 1988). Two of these sibships are also included in the CEPH collaboration (CEPH codes 102 and 104). We have recently expanded our reference set of pedigrees to include 73 sibships now comprising over 1100 potentially informative meioses. There is no overlap with the CEPH in these newer sibships. A subset of markers (D17S71, D17S57, D17S73, D17S33, and D17S58) spanning the region that includes the NFl gene was typed on most of the expanded reference set.
In 1980 it was proposed (Botstein et al.) that restriction fragment length polymorphisms (RFLPs) could be used as reference markers for the generation of genetic linkage maps of entire chromosomes. Since that time, a large number of genetic linkage maps have been published (e.g., Drayna and White, 1985; Donis-Keller et al., 1987; Tanzi et al., 1988). These maps serve a dual purpose. First, they serve as a reference source for determining the best set of markers available for identifying the chromosomal location of human disease genes.Second, these maps can serve as reference points for generating physical maps and comparing genetic and physical distances. Chromosome 17 has engendered considerable interest since the mapping of von Recklinghausen neurofibromatosis (NFl) (Barker et al., 1987; Seizinger et al., 1987) and one form of Charcot-Marie-Tooth disease(CMT2) (Vance et al., 1989) to the pericentromeric region and the identification of the TP53 gene as the probable site of mutations predisposing toward colorectal cancer (Vogelstein et al., 1989; Baker et al., 1989). Several other chromosome 17 maps have been published (Ruano et al., 1988; Nakamura et al., 1988; Goldgar et al., 1989), providing either widely spaced reference points along large portions of the chromosome or
Probes Methods for probe preparation, DNA digestion, Southern blotting, and hybridization have been described in detail elsewhere (Tanzi et al., 1988). Table 1 provides detailed descriptions of each probe. Genetic Linkage Analysis All data were entered into a computer file using the LIPIN (Trofatter et al., 1986) data management pro1 Copyright 0 1990 All rights of reproduction
osss-7543/90 $3.00 by Academic Press, Inc. in any form reserved.
2
HAINES
ET
TABLE Information Prohe pYNZ22.1 pHF12-2
Designation D17S5 D17Sl
p53-H7
TP53
pHRpII5.5 pA1041
POLRP D17S71
LEW301 pHHH202 pEW207
D17S58 D17S33 D17573
LEW206 p9TAU LEW203 HPP pE51
D17S57 MTBTl D17S54 PPY NGFR
pCMM86 chaH800
D17S74 GH
pHtk9
TKl
GAA67 BS3 pTHH59
GAA HOX2 D17S4
on Markers
Enzyme MspI &XI
MspI Haplotype SCUI B&II Haplotype Hind111 MspI PUUII Haplotype Tag RsaI B&I Hind111 Huplotype MspI XbaI B&I MspI HincII XmnI XmnI MspI HincII B&II BglII Hind111 Sea1 BstEII Haplotype Sad Taq
gram. Data can then be modified and converted into LIPED, LINKAGE, and MAPMAKER formats with ease.The sibships were entered as nuclear families (including, where possible, grandparents). This allows the use of more efficient algorithms for analysis and results in little loss of linkage information (Haines, unpublished results). All linkage analyses were performed using the MAPMAKER program (version 1.0) (Lander et al., 1987). Our general procedure is to generate all possible two-point lod scores and examine them to determine a set of three to four markers that appear to be well spaced (5-15 CM apart) and mutually informative. We then examine all possible orders to determine the most likely order, and accept that as definitive if it has odds greater than 1OO:l better than any other order. New markers are then tried in every possible position along the current map and are placed only if the odds of one position are at least 1OO:l better than those of any other position on the map. In this way, we can build a uniquely ordered set of markers. All order determinations were performed using equal recombination fractions in both sexes.
AL.
1
Used for Chromosome Allele
frequency
VNTR 0.69 0.31 0.65 0.35
17 Observed
heterozygosity
Ref.
0.86
(21) (24
0.63
(16) 0.77 0.06 0.17 0.00 0.22 0.78 0.50 0.50 0.19 0.81 0.00 0.61 0.08 0.31 0.40 0.60 0.57 0.43 0.80 0.20 0.75 0.25 0.33 0.45 0.22 0.00 0.30 0.70 0.78 0.22 0.66 0.34 0.52 0.48 0.64 0.34 0.92 0.08 0.02 0.98 VNTR 0.67 0.33 0.25 0.75
0.54 0.46
(31)
(23) 0.50 0.43 0.35
0.60 0.36 0.29 0.42 0.42 0.33
0.90 0.50
(2) 03) (10)
(2) (2’0 (2) (11)
(6) (22) (7)
U&20) 0.34 0.02 0.64 0.03 0.05 0.24 0.68 0.85 0.15 VNTR
0.71 0.42 0.29 0.76
(15) (1%
(21)
Early in this process, we realized that errors in the data could have a profound effect on the ability to place a marker uniquely, as those errors would most likely be detected as double recombination events. To facilitate error checking, we have developed the program CHROMLOOK (Haines, in preparation), which examines the inheritance of each marker (given a particular marker order) and determines the probable maternal or paternal origin of each child’s alleles. The output consists of paternal and maternal alleles for each child, with recombination events marked. Almost all recombination events can be detected in this way. Apparent occurrences of double recombinations were checked against the original autorads for errors, and many such events were removed by entering the corrected data. In a few cases,these events were resolved only when markers were retyped. This extensive error checking allowed us to increase the number of markers with unique positioning from 10 to 13. Sex-specific lod scoreswere calculated after the final map was determined. Differences were tested for significance using the likelihood ratio test. The -2 In likelihood approximates a X2 distribution where the
LINKAGE
MAP
OF
number of degrees of freedom is determined by the difference in the number of parameters estimated. RESULTS
Table 2 presents the peak recombination fractions and lod scores for all possible two-point combinations. These data were used to identify a set of four markers (NGFR, GH, TKl, and GAA) that produced a unique ordering and was the starting set on which the rest of the map was built. The choice of this set of markers was somewhat arbitrary; a similar set of markers elsewhere on the chromosome could have been used instead. Figure 1 presents the sex-averaged and sex-specific maps, along with physical localizations of some of the markers. Three markers could not be placed uniquely within the final map. D17S4 may reside on either side of TKl; its preferred position is between GH and TKl, but the odds are only l&l over a position between TKl and GAA. D17S57 could be on either side of D17S73; the odds are essentially equal for either position. HOX2 could reside in any position from D17S73 distal to D17S74. Its preferred position is between D17S54 and PPY, with odds over the next best position being 16: 1. Since these markers were not uniquely placed, they were not used in the tests of recombinational sex differences. Three loci in the map, TP53, POLRB, and D17S1, are very close to one another, and their relative order could not be determined. In fact, only the orders D17S5-D17Sl-TP53-POLR2-D17S71 and D17S5TP53-D17Sl-POLR2-D17S71 could be rejected at 1OO:l odds (Table 3). Of the 22 triply informative
TABLE Two-Point Marker
S5
55 TP53 POLR2 Sl s71 558 533 573 MTBTl s54 PPY NGFR s74 GH TKl GAA s57 54 HOX2
3.04 5.00 3.92 0.00 0.85 0.12 0.13 0.00 0.03 0.21 0.00 0.47 0.12 0.16 0.13 0.10 0.00 0.00
a Not
TP53 0.28
informative.
16.39 9.05 1.35 1.80 0.14 0.74 1.26 0.76 0.86 2.57 1.96 0.13 0.00 0.14 0.65 0.00 0.03
POLR2 0.24 0.01 15.07 3.66 1.30 0.18 0.13 1.19 0.53 0.09 2.23 1.48 2.82 0.99 0.00 0.48 0.11 0.60
Lod Scores Sl 0.28 0.05 0.03 1.68 0.68 0.00 0.24 0.66 2.20 0.15 2.62 0.62 0.52 0.00 0.00 0.55 0.00 0.00
(Below
Diagonal)
571
S58
s33
573
0.50 0.33 0.24 0.28
0.33 0.20 0.23 0.29 0.08
0.44 0.34 0.39 0.50 0.11 0.03
0.45 0.36 0.42 0.35 0.11 0.05 0.06
21.96 13.65 27.50 0.00 18.37 0.00 3.93 2.68 1.47 0.65 0.00 24.40 0.00 1.02
22.04 25.81 2.84 15.50 3.74 4.63 8.49 3.02 1.31 0.18 12.06 0.39 1.13
19.60 3.24 18.35 0.09 2.58 1.36 0.22 1.85 0.36 17.69 2.58 0.95
3.90 34.43 2.54 3.65 4.53 0.98 0.89 1.01 41.20 0.11 0.51
MTBTl 0.50 0.26 0.16 0.32 0.50 0.14 0.12 0.10
CHROMOSOME
3
17
events, only two crosses were observed, both arguing for the order D17S5-TP53-POLR2-D17Sl-D17S71. However, all three loci are included in the map because each is necessary to obtain more accurate map distances given the distribution of informativeness of these markers relative to the flanking markers D17S5 and D17S71. The overall female to male ratio of recombination fraction along the entire map is 2.25:1. We tested for significance of the sex differences using two different models. If we let each interval vary independently, the ~‘(15) value is 71.42 (P < 0.005), indicating that there is significant variation by sex compared to a null hypothesis of a 1:l sex ratio across the entire map. We then examined the possibility that only one or two regions might be responsible for this difference. We examined the data by defining three regions: D17S5D17S58, D17S58-NGFR, and NGFR-GAA. In each case [X2(5) = 27.4, P < 0.005; x’(6) = 23.67, P < 0.005; x2(4) = 20.35, P < 0.005, respectively] the sex differences are statistically significant compared to a 1:l sex ratio. With one nonsignificant exception (TKl-GAA) all intervals had larger female than male recombination fractions. DISCUSSION
We have generated a linkage map with 13 distinct reference points extending virtually the entire length of chromosome 17. Four of the nine genes we have mapped (TP53, POLRB, MTBTl, and GAA) have not, to our knowledge, been previously placed on a genetic linkage map. The remaining five genes (NGFR, TKl,
2 and Peak S54 0.44 0.32 0.34 0.25 0.11 0.17 0.06 0.02 0.00
PPY 0.42 0.24 0.32 0.34 0.50 0.14 0.42 0.18 0.10 0.19
0 Values NGFR 0.50 0.20 0.20 0.23 0.16 0.10 0.18 0.05 0.09 0.03 0.05
(Above
Diagonal)
574
GH
0.41 0.30 0.32 0.36 0.29 0.17 0.32 0.22 0.11 0.16 0.24 0.12
0.45 0.40 0.24 0.36 0.32 0.20 0.36 0.29 0.17 0.24 0.45 0.20 0.09
TKl 0.45 0.50 0.33 0.50 0.38 0.32 0.28 0.33 0.31 0.31 0.47 0.39 0.29 0.23
GAA
S57
S4
0.43 0.34 0.50 0.50 0.50 0.38 0.36 0.27 0.30 0.40 0.27 0.35 0.29 0.26 0.11
0.42 0.50 0.32 0.33 0.08 0.05 0.03 0.00 0.00 0.02 0.24 0.13 0.24 0.35 0.50 0.50
0.50 0.50 0.43 0.50 0.50 0.38 0.25 0.45 0.25 0.33 0.44 0.27 0.31 0.22 0.01 0.12 0.50
8.43 3.59 7.04 8.96 4.23 0.92 0.49 0.90 2.86
1.32 8.81 7.45 2.07 1.18 0.14 32.96 1.23
5.53 3.18 0.03 0.02 0.50 0.88 0.05
9.57 5.15 0.15 0.07 3.81 2.78
21.53 4.04 1.16 2.98 3.45
5.60 1.35 0.23 6.36
9.06 0.00 36.25
0.00 7.25
0.00
0.30
0.75
0.39
1.20
2.24
0.97
1.00
0.04
0.54
a
HOX2 0.50 0.31 0.00 ‘0.50 0.13 0.07 0.12 0.13 0.00 0.18 0.10 0.00 0.14 0.17 0.17 0.32 0.20, (I
HAINES
/
/
/
ET
/
/
/
/
/
/
/
AL.
31
,,Q I/, '
/// $5 /
'//
'5
'//
'//
46
--N
\
\
' \
\ \
\ \
D17S73 MTBTll 017854
--_ II
-_ --
\
Dl7S50 D17S33
IO
---_ ------>
25
D17S71
- - 3'
_--___---23 24
F
, /. / , / / 17
/
22
D17S5
/
/
--_ - .
HOX2
NGFR
25
'\ \
'\
\
\
\
\
D17S74 17
\
\
GH
\ \ 1
\
\
1
32
\
1
\ '1
\
D17S4 TKl
' \9 \
FIG. mapping mapping
Dl7S57
PPY
,5
\
1
d
t
GAA
?
1. Genetic linkage map of chromosome 17 showing male, sex-averaged, and female maps. Distances are indicated in CM (Kosambi function). Physical mapping of some markers is indicated by the square brackets on the idiogram. Markers with only regional linkage are indicated on the far right; the tick marks show the most preferred positions.
HOX2, PPY, and GH) have never appeared in the same genetic linkage map before. Our map, based on a separate data set, has confirmed the order and, more roughly, the distance estimates of maps based on the CEPH or other pedigrees. Tsipouras et al. (1988) attempted to map GH and COLlAl relative to the centromere, but were unable to generate a unique statistically significant order. Two preliminary reports (Miki et al., 1987; Ruano et al., 1988) have presented data for linkage of HOX2, NGFR, and PPY. Our results are entirely consistent with
theirs. In fact, the distance between PPY and NGFR in both reports (5 CM) is very close to our own estimate of 4 CM. Our inability to localize HOX2 results from its relative lack of heterozygosity in the Venezuelan Reference Pedigree. Ruano et al. (1988) reported no recombination between HOX2 and NGFR in approximately 45 informative events. We also observe no recombination, but have only four mutually informative events. HOX2 was placed proximal to NGFR with odds of 27:l over a distal position by Goldgar et al. (1989). Their 6 estimates indicate that recombination between
LINKAGE
TABLE Orders
and Relative D17S1, Order
D17S5-TP53-POLR2-D17Sl-D17S71 D17S5-D17Sl-POLR2-TP53-D17S71 D17S5-POLR2-TP53-D17Sl-Dl7S71 D17S5-POLR2-D17Sl-TP53-D17S71 D17S5-D17Sl-TP53-POLR2-D17S71 D17S5-TP53-D17Sl-POLR2-D17S71
MAP
OF
3
Likelihoods and POLRZ
for TP53,
Relative log,, (likelihood) 0.00 -0.57 -1.21 -1.28 -2.31
-2.40
HOX2 and NGFR has been observed. This is interesting in light of the report of Bentley et al. (1989) which places HOX2 and NGFR on the same 590-kb fragment. This may indicate an increased rate of recombination in this region, at least in families segregating NFl. The map of Donis-Keller et al. (1987) includes only one marker used in the current map, thus making comparisons between the two impossible. Recently another linkage map of chromosome 17 was published (Nakamura et al., 1988). The current map shares five markers in common, and these are spread across virtually the entire length of the map, facilitating comparison. Not surprisingly, order is consistent between these maps. However, there is some variation in genetic distance as estimated from the two data sets. In particular, the distance between D17S33 and D17S74 (34 CM vs 44 CM) and the distance between D17S74 and D17S4 (34 CM vs 56 CM) are smaller in our data set. The possible reasons for these differences are many and include true underlying variation and differences in methods of manipulating and analyzing the data. However, the most likely explanation is simply that these differences arise from random sampling variation in the selection of the families being studied. Both maps show a significant excess of female recombination throughout the majority of the map, although this is more pronounced in our data set (female/male ratio 2.251 vs 1.54:l). The excess male recombination observed by Nakamura et al. (1988) was observed only at the telomeres, in regions not covered by the current map. The pericentromeric region of this chromosome has received a tremendous amount of attention since the recent mapping of NFl to 17q11.2. Goldgar et al. (1989) have produced a summary map of this region based on data from families segregating NFL The differences between the current map and that one are small and exist only in the estimates of 13.It is interesting to note that Goldgar et ab (1989) observed a ~-CM distance between D17S57 and D17S73, while we have observed only one recombination event in approximately 200 mutually informative meioses. One must be aware of the possibility that the presence of disease mutations
CHROMOSOME
5
17
in the region being studied may have some impact on the resulting recombination fraction estimates. Thus, it is unclear whether this observed difference is the result of random chance or is of underlying biological significance. This map will be useful for refining the genetic position of the NFl gene and for providing distances necessary for using markers in prenatal diagnosis of this disease. Similarly, refining the genetic localization of CMT2 in the pericentromeric region of this chromosome may also make use of these data. In particular, by using an essentially independent data set for generation of our map, it is now possible to combine our data with those of other groups to produce a denser, more refined, and more accurate genetic map of chromosome 17. This refined map will serve as a useful first step toward the eventual goal of providing a complete physical map of this chromosome and, ultimately, toward the eventual sequencing of the human genome. ACKNOWLEDGMENTS We gratefully thank Drs. Y. Nakamura, D. Barker, P. Murphy, T. Takeuchi, F. Ruddle, P.-F. Lin, R. White, D. Wolf, and R. Neve for kind gifts of their probes. S.T. is a postdoctoral fellow of the Arthritis Foundation. This work was supported in part by NIH Grants NS20012 and NS22031.
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mutations
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A., PHILLIPS, J. A., III, MELLITS, SEEBURG, P. H. (1984). Patterns
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