GENOMICS
11,968-973
(19%)
A Linkage Map Spanning the Locus for Diastrophic
Dysplasia (DTD)
JOHANNA H~~STBACKA,* PERTTI SIsToNEN, *# t IMKA KAITILA, $ BARBARA WEIFFENBACH, 5 KENNETH K. KIDD,~~ AND ALBERT DE LA CHAPELLE* *Department of Medical Genetics, University of Helsinki, and FolkhJlsan institute of Genetics, Helsinki, Finland; t Finnish Red Cross Blood Transfusion Service, Helsinki, Finland; *Department of Clinical Genetics, Helsinki University Central Hospital, Helsinki, Finland; Kollaborative Research, Inc., Bedford, Massachusetts; and “Department of Human Genetics, Yale University Medical School, New Haven, Connecticut Received
March
25, 1991;
0 1991 Academic
MATERIALS
INTRODUCTION
Diastrophic dysplasia (DTD, MIM No. 222600; McKusick, 1990) is an autosomal recessive form of skeletal dysplasia (Lamy and Maroteaux, 1960; Walker et aZ., 1972). Patients suffer from shortlimbed growth, leading to a mean adult height of 124 cm in females and 141 cm in males (Kaitila et al., 1989). Most patients are born with atypical clubfeet; about half also have cleft palate. Other symptoms comprise generalized joint dysplasia resulting in limitations of joint movements, and often premature osteoarthrosis particularly in the hips. Spinal deformities are common and a typical deformity of the upper ear lobe is a pathognomonic feature. DTD seems to occur at low prevalence in most populations, but is particularly common in Finland (Kaitila, 1980). The biochemical defect in DTD is not known. Abnormal histopathology and irregular distribution and 968 Inc. reserved.
AND
METHODS
The family series was composed of 16 Finnish families with one to three affected children (Fig. 1). The families consisted of two generations except for two sibships in which no parents were available for study. Altogether 95 individuals including 35 affected were analyzed. A blood sample was collected from each individual. Part of the specimen was used to establish an Epstein-Barr virus-transformed lymphoblast line as a permanent source of material (Anderson and Gusella, 1984) and the rest was used for extraction of high-molecular-weight DNA using standard procedures. The 16 polymorphic markers used in this study and their allele frequencies are described in Table 1. Digestion, electrophoresis, Southern hybridization, and autoradiography were carried out as previously described (Hastbacka et al., 1990). The linkage analyses in the DTD families were performed using the programs ILINK and MLINK (for two-point analyses) and LINKMAP (for multipoint analysis) of the LINKAGE package (Lathrop et aZ., 1984). The fixed map of markers against which the multipoint calculations were carried out was generated by combining the data from the public Centre d’Etude du Polymorphisme Humain (CEPH) version
Press, Inc.
0888-7543/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
July 31, 1991
organization of collagen fibrils observed in diastrophic cartilage (Horton et al., 1979) prompted a search of linkage to the gene encoding collagen II (COLPAl), the most abundant collagen of cartilage. However, linkage between DTD and COL2Al was not detected (Elima et al, 1989). We have recently demonstrated linkage of DTD to polymorphic markers on 5q (Hastbacka et al., 1990). Here we report studies that allowed us to place DTD in a 17.5CM interval of an extended linkage map including the GRL and SPARC genes. Two polymorphic loci showed no recombination with DTD.
Diastrophic dysplasia (DTD) is an autosomal recessive osteochondrodysplasia. Patients have short-limbed short stature and suffer from generalized joint dysplasia. We have recently mapped DTD to the distal long arm of chromosome 5. Here we report the localization of DTD in relation to 16 polymorphic markers on distal 5q. No recombinations occurred with two loci, D5S72 and D5S66. One presumptive candidate gene, osteonectin @PARC), could be excluded on the basis of three recombinations with the DTD locus. Multipoint linkage analysis performed against a fixed order of markers placed DTD between glucocorticoid receptor (GRL) and SPARC favored by the odds of 33:l over the next best location of DTD between D5S72 and D5S55. The sex-averaged distance between the detlnite flanking markers, GRL and DSS55, is 17.5 CM. From previously reported data on the physical localization of markers, we conclude that the DTD locus is in 5q31q34.
revised
A LINKAGE
Family number
1 2 3 4 5 6 7 6
MAP
Mother
Offspring
q q q 0 q q q q
0 0 0 0 0 0 0 0
q ICIOOOUB n O 0.000 l UO.0 l o l mIl l H l WOI
cl
11 12 13 14
THE
Father
Cl cl q
15
DTD
LOCUS
969
l uuRoooooo OH q 000.0.0.0000 000
9 10
SPANNING
0 0 0 0 0 0
l UO moo IO
moo
16
Cl0 Be
Unaffected
FIG. 1.
The
composition
3 database with data produced by us (GRL and SPARC) on the CEPH reference family panel of 40 families and data on 14 DTD families. The marker order and distances were evaluated by the program CILINK, computing first all possible permutations for the markers GRL, SPARC, D5S72, D5S55, and D5S61. The orders with odds greater than 103:1 were excluded from further calculations and remaining orders refined by including markers D5S66 and D5S68. The female/male recombination ratio, distances, and order were determined by using the constant sex difference option of CILINK for calculation of the final seven-point orders of the above markers. The two-point lod scores for the combined data were calculated by the program CLODSCORE. RESULTS The two-point lod scores are shown in Table 2. Two markers, D5S72 and D5S66, did not show any recom-
of the DTD
families
studied.
binations with the DTD locus. The maximum lod score for linkage between DTD and D5S72 was 9.10 at B = 0 and the observed heterozygosity in the DTD parents 0.63. With D5S66 the lod score maximum was 2.32 at fl = 0 and the observed heterozygosity 0.26. The second highest lod score of 8.25 at a recombination fraction of 0.04 was obtained with the haplotype constructed from two polymorphisms of the human osteonectin gene locus (SPARC). Three recombinations were observed. Of the remaining markers, four, FGFA, D5S55, D5S61, D5S22, showed linkage at recombination fractions of 0.10 or less, but the lod scores were lower (between 1.75 and 4.31). The data are available from the authors on request. We also calculated marker versus marker linkage both in the DTD families and the combined DTD and CEPH families (Table 3). As can be seen, locus D5S72 and the SPARC locus showed very close linkage, the lod score being 42.10 at B = 0.02 when the CEPH and DTD families were combined. Likewise D5S61 and
970.
HASTBACKA
ET
TABLE Probes
Locus
Probe
D5S58 D5S52 D5S65 D5S67 D5S70 D5S68 GRL FGFA D5S72 SPARC SPARC D5S55 D5S61
CRI-R379 CRI-L1265 CRI-L401 CRI-L986 pTP5E CRI-L1194 phGR1.2 PMJ23a CRI-P148 pHVON-9-2 pHVON-9-2 CRI-C61 CRI-L45
D5S61 D5S22 D5S66 D5S62 DRDl
CRI-L45 pJ0205H-C CRI-P152 CRI-L1200 HG Dl-ES2.9.
’ Number
Enzyme
Alleles
MspI TaqI TaqI Td TaqI MspI BclI EcoRI TwI Td
1
Allele
Freauencies Reported allele frequencies
(kb)
23, 16.5 and 5.0, 7.2 and 5.0, 12 10, 7.8, 7.5, 6.7 9.8, 7.2 and 2.75 12.0, 6.6 and 6.2 12.5, 5.0 16.5, 15.0 4.5, 2.3 3.2, 2.4, 1.0 12.3, 11.5, 9.5 5.2, 4.9,3.3 4.6, 2.9 7.8, 6.1, 5.0, 4.8 7.4, 4.0 and 3.0, 3.8 and 3.2, 4.0 and 2.8 12.9, 8.1 and 4.8 7.0, 4.6, 4.4 9.8, 9.1 3.2, 2.9, 2.7, 2.5 10.5, 6.8
MspI TwI MspI TanI MspI BglII TaqI EcoRI
of chromosomes
and Their
AL.
Allele frequencies observed in DTD parents
0.60/0.14/0.18/0.08 0.17/0.53/0.29/0.01 0.7510.25 0.32/O&3 0.83/0.17 0.20/0.80 0.55/0.45 0.12/0X3/0.05 0.30/0.51/0.19 0.72/0.14/0.14 0.43/0.57 0.47/0.14/0.38/0.01 0.38/0.37/0.23/0.02
0.85/0.06/0.07/0.02 0.09/0.61/0.30/0.00 0.83/0.17 0.50/0.50 0.91/0.09 0.18/0.82 0.4610.54 0.10/0.81/0.09 0.33/0.48/0.19 0.84/0.10/0.06 0.4210.58 0.44/0.15/0.41/0.00 0.37/0.23/0.40/0.00
0.37/0.33/0.30 0.7510.25 0.14/0.82/0.01/0.03 0.28/0.72
0.67/0.33 0.37/0.23/0.40 0.7810.22 0.00/0.13/0.02/0.85 0.9fO.91
no
Ref.
(46)
(48) (52)
(3,281 (3,28) (3, 28) (3,28) (5) (3,28) (10, 18) (9) (3,28) (19) (19) (3, 28) (3, 28)
(42)
(3)
(56) (46) (46) (46) (50)
(46) (48) (52)
(52) (5%
(52)
(mm
(50) (46)
(3,28) (3, 28)
(46)
(22)
scored.
D5S55 are tightly linked with a lod score of 40.65 at ~9 = 0.02. The recombination fractions obtained from the DTD families alone did not differ notably from those obtained from the combined families. To obtain a fixed map of marker loci containing all the information available we used the combined data from the CEPH public database and data generated by us from 31 CEPH families informative for SPARC, 10 CEPH families informative for GRL, and 14 DTD
TABLE Two-Point
Lod Scores
between
families for each of the seven markers used in the estimation of the most likely fixed map. The map used in the multipoint linkage analysis is shown in Fig. 2. It was derived from the best seven-point order of loci, which was ten-(D5S68, GRL)-SPARC-D5S72D5S55-D5S61-D5S66ter. The map shown can be replaced with one that inverts the loci ‘SPARC and D5S72 with the odds of 811. This inversion would result in a most likely location of DTD proximal to
2 DTD
and Marker
Loci
Locus
0.00
0.001
0.01
0.05
0.10
0.20
0.30
GlU
LX
95% Confidence limits
D5S58 D5S65 D5S67 D5S70 D5S52 D5S68 GRL FGFA D5S72 SPARC D5S55 D5S61 D5S22 D5S66 D5S62 DRDl
-00 -co -cc -cc -00 -00 -co
-1.40 -5.56 -9.75 -4.20 -7.76 -1.14 -5.55 0.37 9.08 4.64 -2.55 -3.36 -3.95 2.31 -8.99 -3.95
-0.43 -2.61 -4.81 -2.22 -2.89 -0.17 -1.65 1.31 8.88 7.36 0.33 1.43 0.85 2.27 -5.02 -1.98
0.16 -0.74 -1.61 -0.93 0.01 0.37 0.68 1.74 7.99 8.23 1.91 3.99 3.45 2.08 -2.37 -0.71
0.31 -0.12 -0.50 -0.46 0.78 0.47 1.28 1.69 6.86 7.56 2.15 4.30 3.82 1.80 -1.35 -0.27
0.31 0.22 0.18 -0.12 0.88 0.40 1.25 1.27 4.57 5.31 1.72 3.38 3.04 1.20 -0.51 0.00
0.19 0.19 0.22 -0.02 0.53 0.22 0.76 0271 2.39 2.84 0.97 1.91 1.72 0.61 -0.17 0.04
0.34 0.23 0.24 0.00 0.95 0.48 1.37 1.75 9.10 8.25 2.15 4.31 3.82 2.32 0.00 0.04
0.14 0.23 0.26 0.50 0.16 0.11 0.14 0.06 0.00 0.04 0.10 0.09 0.10 0.00 0.50 0.29
0.00-0.05 0.01-0.10 0.03-0.19 0.03-0.20 -
i1”o -Cc -al -00 -co 2.32 -cc -co
A LINKAGE
TABLE
Maximum
D5S61 D5S55 SPARC D5S72 GRL
SPANNING
3
Lod Scores of Linkage Versus Marker
Pairwise
MAP
of Marker
lod score/g,,
GRL
D5S72
SPARC
D5S55
D5S61
3.80 (5.69) 4.52 (8.19) 3.93 (6.06) 2.94 (10.27) -
10.32 (31.43) 6.95 (18.14) 17.13 (42.10) -
12.14 (29.60) 9.04 (21.94) -
8.43 (40.65) -
-
0.12 (0.11)
0.01 (0.02) 0.13 (0.14)
0.02 (0.08) 0.02 (0.07) 0.08 (0.09)
(X::) 0.05 (0.09) 0.05 (0.07) 0.07 (0.19)
Note. The maximum lod scores are given above the diagonal line and the corresponding B maxima below the diagonal line. Values given without parentheses are for DTD and within parentheses from a combination of DTD and CEPH data.
D5S72 (data not shown). Using the program LINKMAP a multipoint linkage analysis was performed by running DTD against the best fixed order of four markers. The maximum multipoint lod score was 10.47 with the peak at 4.21 CM distal of GRL. This is shown in Fig. 2. The likelihoods of alternative locations of DTD are given in the legend to Fig. 2. The next best location of DTD (8.53 CM distal of GRL) is 33 times less likely. The distances refer to male map distances. On the basis of published data of the physical locations of GRL and the marker D5S22, which flank DTD, we conclude that the DTD locus resides in 5q31-q34 (Giuffra et al., 1988; Theriault et aZ., 1989; Wasmuth et al., 1989; Weiffenbach et al., 1991). Moreover, SPARC has been assigned to 5q31-q33 (Swaroop et al., 1988). DISCUSSION
The ultimate goal of our research is to clone and characterize the DTD gene. At present, we are not aware of obvious candidate genes. Recessively inherited disorders are probably less likely than dominant ones to be caused by mutations in genes encoding structural proteins such as collagen. This is borne out in osteogenesis imperfecta, where dominantly inherited forms of the disease are due to mutations in collagen genes, whereas this does not appear to be the case in recessive forms (Sykes et aZ., 1990). In diastrophic dysplasia cartilage and connective tissue appear to be the only tissues affected (Horton et al., 1979). While this might be taken to indicate that DTD should be found among only a limited number of can-
THE
DTD
LOCUS
971
didate genes, it hardly offers any promising clue to the nature or function of the gene. For cartilage and bone to develop normally a large number of metabolic pathways must be undisrupted, so that many structural and regulatory genes are involved. For these reasons we are pursuing the goal of identifying the DTD gene by first mapping it as precisely as possible. In the present paper weehave narrowed the interval in which DTD resides to that between GRL and D5555. This region has a genetic length, of some 17.5 CM on our sex-averaged map, so it is still too large for efficient physical mapping. However, if the position of DTD between GRL and D5S72 as suggested but not finally proven by our present findings can be confirmed, the interval is narrowed to about 8 CM. We are therefore currently working on refining the map and establishing a definite gene order through linkage analysis
L 0
8.
a
-41 -20
-1.5
-10
-5
’
0
,‘,
5
10
L
15
20
’
25
CM FIG.
2.
Multipoint linkage map of the DTD versus four polymorphic chromosome 5q loci. The map was constructed assuming a fixed order of GRL-SPARC-D5S72-D5S55 with the distances and order estimated from combined DTD and CEPH family data by program CILINK using seven marker loci (see text). The marker location for GRL was selected as starting point (at zero), fixing the other locations at 6.03 for SPARC, at 7.03 for D5S72, and at 11.05 for D5S55 using Kosambi mapping function. Lod sores were computed by the program LINKMAP of the LINKAGE package (Ref. (16)) by selecting the constant sex difference option of female/male distances set at 1.8. The distances shown on the X-axis refer to male map distances. Lod score was calculated as the difference of log,, likelihood of the DTD locus being at a given location on the map versus it being at an infinite distance (0 = 0.50) from the map. The location of the peaks, their corresponding lod scores (Z), and odds compared to the best supported location were (from left to right) as follows: -5.02 CM, 2 = 6.162, odds 2037o:l; 4.21 CM, Z = 10.471, odds 1:l; 6.78 CM, Z = 7.122, odds 2234:l; 8.53 CM, Z = 8.946, odds 33:l; and 14.55 CM, Z = 7.259, odds 1629:l. S55 and S72 are abbreviations for D5S55 and D5S72, respectively.
HASTBACKA
912
with new markers from the area (Bishop and Westbrook, 1990). Once DTD can be assigned to an interval of about 1 CM, it can be searched for more efficiently by physical mapping and other methods. Using a strategy involving cells from patients with acquired deletions of 5q, somatic cell hybrids retaining fragments of chromosome 5, Southern hybridization, and pulsed-field gel electrophoresis, Huebner et al. (1990) constructed a map of chromosome 5. The suggested order in distal 5q was ten-GMCSFFGFA-(CSFlR, PDGFR)-(treC, ADRBR)-(ARHH9, CSFl)-ter. The map position of GRL could not be unequivocally determined but the best estimate consistently placed it telomeric of GMCSF and centromeric of the pair (CSFlR, PDGFR) in the vicinity of FGFA. The latter pair is known to be tandemly organized lessthan 0.5 kb apart (Roberts et al., 1988). It is desirable to be able to superimpose the physical map of Huebner et al. (1990) on our linkage map. At present, the only markers used in both studies are GRL and FGFA. This does not provide sufficient information to reliably integrate the maps. We anticipate placing as many as possible of the gene loci that are assigned to the region on our linkage map. This will allow us to establish or rule them out as putative candidate genes for DTD. In this paper we show that one possible candidate gene, osteonectin @PARC locus), can be ruled out on the basis of at least three recombinations with DTD in our panel of affected families. That osteonectin is not the putative DTD gene is supported not only by these recombinations but also by the fact that DTD cosegregated with three of four allele haplotypes of SPARC detected in DTD families (data not shown). Because DTD is so clearly enriched in the isolated Finnish population, we presume that many of the patients have the same mutation; hence strong linkage disequilibrium is likely with very close markers. As noted by Bishop and Westbrook (1990), receptor genes continue to be localized to 5q with surprising consistency. Recent additions include the human dopamine Dl receptor (Grandy et al., 1990; Sunahara et al., 1990) and some of the adrenergic receptor genes (Yang-Feng et al., 1990). It remains to be established exactly where DTD maps with respect to those receptor genes; it should not be overlooked that DTD might itself encode a receptor. ACKNOWLEDGMENTS Our thanks are due to Dr. Ronald M. Evans for the probe phGR1.2, Drs. George L. Long and Susan Naylor for the probe pHVON-9-2, Dr. Michael Jaye for the probe PMJ3la, Dr. Hyman B. Niznik for the probe HGDl-ES2.9., and Collaborative I&search, Inc., for the probes CRI-L1265, CRI-C61, CRI-R379, CRIL45, CRI-L1200, CRI-L401, CRI-P152, CRIL986, CRI-L1194, and CRI-P148. Probes pJ0205H-C and pTP5E were obtained
ET AL. from ATCC, where they have been deposited by Drs. John Wasmuth and Rosann Farber, respectively. We thank Ms. Elvi Karila and Ms. Ann-Liz Trsskelin for technical assistance and Ms. Sinikka Lindh for collecting the samples. This study has been supported by grants from the Finska Liikaresallskapet, the Sigrid Juselius Foundation, the Academy of Finland, and National Institutes of Health.
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A LINKAGE
14.
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18. 19.
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Structure and Genetic Disorders” (A. W. Eriksson, H. Forsius, H. R. Nevanlinna, P. L. Workman, and R. K. Norio, Eds.), pp. 610-613, Academic Press, London. KAITILA, I., MARITINEN, E., MERIKANTO, J., POUSSA, M., AND RY~PPY, S. (1989). Clinical expression and course of diastrophic dysplasia. Am. J. Med. Genet. 34: 141. LAMY, M., AND MAROTEAIJX, P. (1960). Le nanisme diastrophique. Presse Med. 68: 1977-1986. LATHROP, G. M., LALOUEL, J. M., JULIER, C., AND Om, J. (1984). Strategies for multilocus linkage analysis in humans. Proc. Natl. Acad. Sci. USA 81: 3443-3446. MCKUSICK, V. A. (1990). “Mendelian Inheritance in Man: Catalogs of Autosomal Dominant, Autosomai Recessive, and X-Linked Phenotypes,” 9th ed., p. 1142, Johns Hopkins Univ. Press, Baltimore, MD. MURRAY, J. C., SMITH, R. F., ARDINGER, H. A., AND WEINBERGER, C. (1987). RFLP for the glucocorticoid receptor (GRL) located at 5qll-ql3. Nucleic Acids Res. 16: 6765. NAYLOR, S. L., HELEN-DAVIS, D., CHAROENWOFIAWAT, P., VILLAREAL, X. C., AND LONG, G. L. (1989). The human osteonectin gene (OSN) has TaqI and MspI polymorphisms. Nucleic Acids Res. 17: 6753. OVERHAUSER, J., MCMAHAN, J., AND WASMUTH, J. J. (1987). Identification of 28 DNA fragments that detect RFLPs in 13 distinct physical regions of the short arm of chromosome 5. Nucleic Acids Res. 16: 4617-4627. ROBERTS, W. M., LOOK, A. T., ROUSSEL, M. F., AND SHERR, C. J. (1988). Tandem linkage of human CSF-1 receptor (cfms) and PDGF receptor genes. Cell 55: 655-661. SUNAHAF~A,R. K., NIZNIK, H. B., WEINER, D. M., STORMAN, T. M., BRANN, M. R., KENNEDY, J. L., GELERTNER, J. E., ROZMAHELL, R., YANG, Y., ISRAEL, Y., SEEMAN, P., AND
23.
24.
25.
26. 27.
28.
29.
THE
DTD LOCUS
973
O’DO~D, B. F. (1990). Human dopamine D, receptor encoded by an intronless gene on chromosome 5. Nature (London) 347: 80-83. SWAROOP, A., HOGAN, B. L. M., AND FRANCKE, U. (1988). Molecular analysis of the cDNA for human SPARC/Osteonectin/BM-4O: Sequence, expression and localization of the gene to chromosome 5q31-33. Genomics 2: 37-47. SYKES, B., OGILIVE, D., WORDSWORTH, P., WALLIS, G., MATHEW, C., BEIGHTON, P., NICHOLLS, A., POPE, M., THOMPSON, E., TSIPOU~~AS, P., SCHWARTZ, R., JENS~ON, O., ARNASON, A., BQERRESEN, A.-L., HEIBERG, A., FREY, D., AND STEINMANN, B. (1990). Consistent linkage of dominantly inherited osteogenesis imperfecta to the type 1 collagen loci: COLlAl and COLlA2. Am. J. Hum. Genet. 48: 293-307. THERIAULT, A., BOM, E., HARRAP, S. B., HOLLE~ERG, S. M., AND CONNOR, J. M. (1989). Regional chromosomal assignment of the human glucocorticoid receptor gene to 5q31. Hum. Genet. 83: 289-291. WALKER, B. A., SCOTT, C. I., HALL, J. G., MURDOCH, J. L., AND MCKUSICK, V. A. (1972). Diastrophic dwarfism. Medicine 51: 41-59. WASMUTH, J. J., PARK, C., AND FERRELL, R. E. (1989). Report of the committee on the genetic constitution of Chromosome 5. Cytogenet. Cell Genet. 61: 137-148. WEIFFENBACH, B., FALLS, K., BRICKER, A., HALL, L., McMAHON, J., WASMUTH, J., FUNANAGE, V., AND DONIS-KELLER, H. (1991). A genetic linkage map of human chromosome 5 with 60 RFLP loci. Gerwmics 10: 173-185. YANG-FENG, T. L., Xm, F., ZHONG, W., COTECCHU, S., FRIELL, T., CARON, M., L~~~owrrz, R. J., AND FRANC=, U. (1990). Chromosomal organization of adrenergic receptor genes. Proc. Natl. Acad. Sci. USA 87: 1516-1520.
11,968-973
(19%)
A Linkage Map Spanning the Locus for Diastrophic
Dysplasia (DTD)
JOHANNA H~~STBACKA,* PERTTI SIsToNEN, *# t IMKA KAITILA, $ BARBARA WEIFFENBACH, 5 KENNETH K. KIDD,~~ AND ALBERT DE LA CHAPELLE* *Department of Medical Genetics, University of Helsinki, and FolkhJlsan institute of Genetics, Helsinki, Finland; t Finnish Red Cross Blood Transfusion Service, Helsinki, Finland; *Department of Clinical Genetics, Helsinki University Central Hospital, Helsinki, Finland; Kollaborative Research, Inc., Bedford, Massachusetts; and “Department of Human Genetics, Yale University Medical School, New Haven, Connecticut Received
March
25, 1991;
0 1991 Academic
MATERIALS
INTRODUCTION
Diastrophic dysplasia (DTD, MIM No. 222600; McKusick, 1990) is an autosomal recessive form of skeletal dysplasia (Lamy and Maroteaux, 1960; Walker et aZ., 1972). Patients suffer from shortlimbed growth, leading to a mean adult height of 124 cm in females and 141 cm in males (Kaitila et al., 1989). Most patients are born with atypical clubfeet; about half also have cleft palate. Other symptoms comprise generalized joint dysplasia resulting in limitations of joint movements, and often premature osteoarthrosis particularly in the hips. Spinal deformities are common and a typical deformity of the upper ear lobe is a pathognomonic feature. DTD seems to occur at low prevalence in most populations, but is particularly common in Finland (Kaitila, 1980). The biochemical defect in DTD is not known. Abnormal histopathology and irregular distribution and 968 Inc. reserved.
AND
METHODS
The family series was composed of 16 Finnish families with one to three affected children (Fig. 1). The families consisted of two generations except for two sibships in which no parents were available for study. Altogether 95 individuals including 35 affected were analyzed. A blood sample was collected from each individual. Part of the specimen was used to establish an Epstein-Barr virus-transformed lymphoblast line as a permanent source of material (Anderson and Gusella, 1984) and the rest was used for extraction of high-molecular-weight DNA using standard procedures. The 16 polymorphic markers used in this study and their allele frequencies are described in Table 1. Digestion, electrophoresis, Southern hybridization, and autoradiography were carried out as previously described (Hastbacka et al., 1990). The linkage analyses in the DTD families were performed using the programs ILINK and MLINK (for two-point analyses) and LINKMAP (for multipoint analysis) of the LINKAGE package (Lathrop et aZ., 1984). The fixed map of markers against which the multipoint calculations were carried out was generated by combining the data from the public Centre d’Etude du Polymorphisme Humain (CEPH) version
Press, Inc.
0888-7543/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
July 31, 1991
organization of collagen fibrils observed in diastrophic cartilage (Horton et al., 1979) prompted a search of linkage to the gene encoding collagen II (COLPAl), the most abundant collagen of cartilage. However, linkage between DTD and COL2Al was not detected (Elima et al, 1989). We have recently demonstrated linkage of DTD to polymorphic markers on 5q (Hastbacka et al., 1990). Here we report studies that allowed us to place DTD in a 17.5CM interval of an extended linkage map including the GRL and SPARC genes. Two polymorphic loci showed no recombination with DTD.
Diastrophic dysplasia (DTD) is an autosomal recessive osteochondrodysplasia. Patients have short-limbed short stature and suffer from generalized joint dysplasia. We have recently mapped DTD to the distal long arm of chromosome 5. Here we report the localization of DTD in relation to 16 polymorphic markers on distal 5q. No recombinations occurred with two loci, D5S72 and D5S66. One presumptive candidate gene, osteonectin @PARC), could be excluded on the basis of three recombinations with the DTD locus. Multipoint linkage analysis performed against a fixed order of markers placed DTD between glucocorticoid receptor (GRL) and SPARC favored by the odds of 33:l over the next best location of DTD between D5S72 and D5S55. The sex-averaged distance between the detlnite flanking markers, GRL and DSS55, is 17.5 CM. From previously reported data on the physical localization of markers, we conclude that the DTD locus is in 5q31q34.
revised
A LINKAGE
Family number
1 2 3 4 5 6 7 6
MAP
Mother
Offspring
q q q 0 q q q q
0 0 0 0 0 0 0 0
q ICIOOOUB n O 0.000 l UO.0 l o l mIl l H l WOI
cl
11 12 13 14
THE
Father
Cl cl q
15
DTD
LOCUS
969
l uuRoooooo OH q 000.0.0.0000 000
9 10
SPANNING
0 0 0 0 0 0
l UO moo IO
moo
16
Cl0 Be
Unaffected
FIG. 1.
The
composition
3 database with data produced by us (GRL and SPARC) on the CEPH reference family panel of 40 families and data on 14 DTD families. The marker order and distances were evaluated by the program CILINK, computing first all possible permutations for the markers GRL, SPARC, D5S72, D5S55, and D5S61. The orders with odds greater than 103:1 were excluded from further calculations and remaining orders refined by including markers D5S66 and D5S68. The female/male recombination ratio, distances, and order were determined by using the constant sex difference option of CILINK for calculation of the final seven-point orders of the above markers. The two-point lod scores for the combined data were calculated by the program CLODSCORE. RESULTS The two-point lod scores are shown in Table 2. Two markers, D5S72 and D5S66, did not show any recom-
of the DTD
families
studied.
binations with the DTD locus. The maximum lod score for linkage between DTD and D5S72 was 9.10 at B = 0 and the observed heterozygosity in the DTD parents 0.63. With D5S66 the lod score maximum was 2.32 at fl = 0 and the observed heterozygosity 0.26. The second highest lod score of 8.25 at a recombination fraction of 0.04 was obtained with the haplotype constructed from two polymorphisms of the human osteonectin gene locus (SPARC). Three recombinations were observed. Of the remaining markers, four, FGFA, D5S55, D5S61, D5S22, showed linkage at recombination fractions of 0.10 or less, but the lod scores were lower (between 1.75 and 4.31). The data are available from the authors on request. We also calculated marker versus marker linkage both in the DTD families and the combined DTD and CEPH families (Table 3). As can be seen, locus D5S72 and the SPARC locus showed very close linkage, the lod score being 42.10 at B = 0.02 when the CEPH and DTD families were combined. Likewise D5S61 and
970.
HASTBACKA
ET
TABLE Probes
Locus
Probe
D5S58 D5S52 D5S65 D5S67 D5S70 D5S68 GRL FGFA D5S72 SPARC SPARC D5S55 D5S61
CRI-R379 CRI-L1265 CRI-L401 CRI-L986 pTP5E CRI-L1194 phGR1.2 PMJ23a CRI-P148 pHVON-9-2 pHVON-9-2 CRI-C61 CRI-L45
D5S61 D5S22 D5S66 D5S62 DRDl
CRI-L45 pJ0205H-C CRI-P152 CRI-L1200 HG Dl-ES2.9.
’ Number
Enzyme
Alleles
MspI TaqI TaqI Td TaqI MspI BclI EcoRI TwI Td
1
Allele
Freauencies Reported allele frequencies
(kb)
23, 16.5 and 5.0, 7.2 and 5.0, 12 10, 7.8, 7.5, 6.7 9.8, 7.2 and 2.75 12.0, 6.6 and 6.2 12.5, 5.0 16.5, 15.0 4.5, 2.3 3.2, 2.4, 1.0 12.3, 11.5, 9.5 5.2, 4.9,3.3 4.6, 2.9 7.8, 6.1, 5.0, 4.8 7.4, 4.0 and 3.0, 3.8 and 3.2, 4.0 and 2.8 12.9, 8.1 and 4.8 7.0, 4.6, 4.4 9.8, 9.1 3.2, 2.9, 2.7, 2.5 10.5, 6.8
MspI TwI MspI TanI MspI BglII TaqI EcoRI
of chromosomes
and Their
AL.
Allele frequencies observed in DTD parents
0.60/0.14/0.18/0.08 0.17/0.53/0.29/0.01 0.7510.25 0.32/O&3 0.83/0.17 0.20/0.80 0.55/0.45 0.12/0X3/0.05 0.30/0.51/0.19 0.72/0.14/0.14 0.43/0.57 0.47/0.14/0.38/0.01 0.38/0.37/0.23/0.02
0.85/0.06/0.07/0.02 0.09/0.61/0.30/0.00 0.83/0.17 0.50/0.50 0.91/0.09 0.18/0.82 0.4610.54 0.10/0.81/0.09 0.33/0.48/0.19 0.84/0.10/0.06 0.4210.58 0.44/0.15/0.41/0.00 0.37/0.23/0.40/0.00
0.37/0.33/0.30 0.7510.25 0.14/0.82/0.01/0.03 0.28/0.72
0.67/0.33 0.37/0.23/0.40 0.7810.22 0.00/0.13/0.02/0.85 0.9fO.91
no
Ref.
(46)
(48) (52)
(3,281 (3,28) (3, 28) (3,28) (5) (3,28) (10, 18) (9) (3,28) (19) (19) (3, 28) (3, 28)
(42)
(3)
(56) (46) (46) (46) (50)
(46) (48) (52)
(52) (5%
(52)
(mm
(50) (46)
(3,28) (3, 28)
(46)
(22)
scored.
D5S55 are tightly linked with a lod score of 40.65 at ~9 = 0.02. The recombination fractions obtained from the DTD families alone did not differ notably from those obtained from the combined families. To obtain a fixed map of marker loci containing all the information available we used the combined data from the CEPH public database and data generated by us from 31 CEPH families informative for SPARC, 10 CEPH families informative for GRL, and 14 DTD
TABLE Two-Point
Lod Scores
between
families for each of the seven markers used in the estimation of the most likely fixed map. The map used in the multipoint linkage analysis is shown in Fig. 2. It was derived from the best seven-point order of loci, which was ten-(D5S68, GRL)-SPARC-D5S72D5S55-D5S61-D5S66ter. The map shown can be replaced with one that inverts the loci ‘SPARC and D5S72 with the odds of 811. This inversion would result in a most likely location of DTD proximal to
2 DTD
and Marker
Loci
Locus
0.00
0.001
0.01
0.05
0.10
0.20
0.30
GlU
LX
95% Confidence limits
D5S58 D5S65 D5S67 D5S70 D5S52 D5S68 GRL FGFA D5S72 SPARC D5S55 D5S61 D5S22 D5S66 D5S62 DRDl
-00 -co -cc -cc -00 -00 -co
-1.40 -5.56 -9.75 -4.20 -7.76 -1.14 -5.55 0.37 9.08 4.64 -2.55 -3.36 -3.95 2.31 -8.99 -3.95
-0.43 -2.61 -4.81 -2.22 -2.89 -0.17 -1.65 1.31 8.88 7.36 0.33 1.43 0.85 2.27 -5.02 -1.98
0.16 -0.74 -1.61 -0.93 0.01 0.37 0.68 1.74 7.99 8.23 1.91 3.99 3.45 2.08 -2.37 -0.71
0.31 -0.12 -0.50 -0.46 0.78 0.47 1.28 1.69 6.86 7.56 2.15 4.30 3.82 1.80 -1.35 -0.27
0.31 0.22 0.18 -0.12 0.88 0.40 1.25 1.27 4.57 5.31 1.72 3.38 3.04 1.20 -0.51 0.00
0.19 0.19 0.22 -0.02 0.53 0.22 0.76 0271 2.39 2.84 0.97 1.91 1.72 0.61 -0.17 0.04
0.34 0.23 0.24 0.00 0.95 0.48 1.37 1.75 9.10 8.25 2.15 4.31 3.82 2.32 0.00 0.04
0.14 0.23 0.26 0.50 0.16 0.11 0.14 0.06 0.00 0.04 0.10 0.09 0.10 0.00 0.50 0.29
0.00-0.05 0.01-0.10 0.03-0.19 0.03-0.20 -
i1”o -Cc -al -00 -co 2.32 -cc -co
A LINKAGE
TABLE
Maximum
D5S61 D5S55 SPARC D5S72 GRL
SPANNING
3
Lod Scores of Linkage Versus Marker
Pairwise
MAP
of Marker
lod score/g,,
GRL
D5S72
SPARC
D5S55
D5S61
3.80 (5.69) 4.52 (8.19) 3.93 (6.06) 2.94 (10.27) -
10.32 (31.43) 6.95 (18.14) 17.13 (42.10) -
12.14 (29.60) 9.04 (21.94) -
8.43 (40.65) -
-
0.12 (0.11)
0.01 (0.02) 0.13 (0.14)
0.02 (0.08) 0.02 (0.07) 0.08 (0.09)
(X::) 0.05 (0.09) 0.05 (0.07) 0.07 (0.19)
Note. The maximum lod scores are given above the diagonal line and the corresponding B maxima below the diagonal line. Values given without parentheses are for DTD and within parentheses from a combination of DTD and CEPH data.
D5S72 (data not shown). Using the program LINKMAP a multipoint linkage analysis was performed by running DTD against the best fixed order of four markers. The maximum multipoint lod score was 10.47 with the peak at 4.21 CM distal of GRL. This is shown in Fig. 2. The likelihoods of alternative locations of DTD are given in the legend to Fig. 2. The next best location of DTD (8.53 CM distal of GRL) is 33 times less likely. The distances refer to male map distances. On the basis of published data of the physical locations of GRL and the marker D5S22, which flank DTD, we conclude that the DTD locus resides in 5q31-q34 (Giuffra et al., 1988; Theriault et aZ., 1989; Wasmuth et al., 1989; Weiffenbach et al., 1991). Moreover, SPARC has been assigned to 5q31-q33 (Swaroop et al., 1988). DISCUSSION
The ultimate goal of our research is to clone and characterize the DTD gene. At present, we are not aware of obvious candidate genes. Recessively inherited disorders are probably less likely than dominant ones to be caused by mutations in genes encoding structural proteins such as collagen. This is borne out in osteogenesis imperfecta, where dominantly inherited forms of the disease are due to mutations in collagen genes, whereas this does not appear to be the case in recessive forms (Sykes et aZ., 1990). In diastrophic dysplasia cartilage and connective tissue appear to be the only tissues affected (Horton et al., 1979). While this might be taken to indicate that DTD should be found among only a limited number of can-
THE
DTD
LOCUS
971
didate genes, it hardly offers any promising clue to the nature or function of the gene. For cartilage and bone to develop normally a large number of metabolic pathways must be undisrupted, so that many structural and regulatory genes are involved. For these reasons we are pursuing the goal of identifying the DTD gene by first mapping it as precisely as possible. In the present paper weehave narrowed the interval in which DTD resides to that between GRL and D5555. This region has a genetic length, of some 17.5 CM on our sex-averaged map, so it is still too large for efficient physical mapping. However, if the position of DTD between GRL and D5S72 as suggested but not finally proven by our present findings can be confirmed, the interval is narrowed to about 8 CM. We are therefore currently working on refining the map and establishing a definite gene order through linkage analysis
L 0
8.
a
-41 -20
-1.5
-10
-5
’
0
,‘,
5
10
L
15
20
’
25
CM FIG.
2.
Multipoint linkage map of the DTD versus four polymorphic chromosome 5q loci. The map was constructed assuming a fixed order of GRL-SPARC-D5S72-D5S55 with the distances and order estimated from combined DTD and CEPH family data by program CILINK using seven marker loci (see text). The marker location for GRL was selected as starting point (at zero), fixing the other locations at 6.03 for SPARC, at 7.03 for D5S72, and at 11.05 for D5S55 using Kosambi mapping function. Lod sores were computed by the program LINKMAP of the LINKAGE package (Ref. (16)) by selecting the constant sex difference option of female/male distances set at 1.8. The distances shown on the X-axis refer to male map distances. Lod score was calculated as the difference of log,, likelihood of the DTD locus being at a given location on the map versus it being at an infinite distance (0 = 0.50) from the map. The location of the peaks, their corresponding lod scores (Z), and odds compared to the best supported location were (from left to right) as follows: -5.02 CM, 2 = 6.162, odds 2037o:l; 4.21 CM, Z = 10.471, odds 1:l; 6.78 CM, Z = 7.122, odds 2234:l; 8.53 CM, Z = 8.946, odds 33:l; and 14.55 CM, Z = 7.259, odds 1629:l. S55 and S72 are abbreviations for D5S55 and D5S72, respectively.
HASTBACKA
912
with new markers from the area (Bishop and Westbrook, 1990). Once DTD can be assigned to an interval of about 1 CM, it can be searched for more efficiently by physical mapping and other methods. Using a strategy involving cells from patients with acquired deletions of 5q, somatic cell hybrids retaining fragments of chromosome 5, Southern hybridization, and pulsed-field gel electrophoresis, Huebner et al. (1990) constructed a map of chromosome 5. The suggested order in distal 5q was ten-GMCSFFGFA-(CSFlR, PDGFR)-(treC, ADRBR)-(ARHH9, CSFl)-ter. The map position of GRL could not be unequivocally determined but the best estimate consistently placed it telomeric of GMCSF and centromeric of the pair (CSFlR, PDGFR) in the vicinity of FGFA. The latter pair is known to be tandemly organized lessthan 0.5 kb apart (Roberts et al., 1988). It is desirable to be able to superimpose the physical map of Huebner et al. (1990) on our linkage map. At present, the only markers used in both studies are GRL and FGFA. This does not provide sufficient information to reliably integrate the maps. We anticipate placing as many as possible of the gene loci that are assigned to the region on our linkage map. This will allow us to establish or rule them out as putative candidate genes for DTD. In this paper we show that one possible candidate gene, osteonectin @PARC locus), can be ruled out on the basis of at least three recombinations with DTD in our panel of affected families. That osteonectin is not the putative DTD gene is supported not only by these recombinations but also by the fact that DTD cosegregated with three of four allele haplotypes of SPARC detected in DTD families (data not shown). Because DTD is so clearly enriched in the isolated Finnish population, we presume that many of the patients have the same mutation; hence strong linkage disequilibrium is likely with very close markers. As noted by Bishop and Westbrook (1990), receptor genes continue to be localized to 5q with surprising consistency. Recent additions include the human dopamine Dl receptor (Grandy et al., 1990; Sunahara et al., 1990) and some of the adrenergic receptor genes (Yang-Feng et al., 1990). It remains to be established exactly where DTD maps with respect to those receptor genes; it should not be overlooked that DTD might itself encode a receptor. ACKNOWLEDGMENTS Our thanks are due to Dr. Ronald M. Evans for the probe phGR1.2, Drs. George L. Long and Susan Naylor for the probe pHVON-9-2, Dr. Michael Jaye for the probe PMJ3la, Dr. Hyman B. Niznik for the probe HGDl-ES2.9., and Collaborative I&search, Inc., for the probes CRI-L1265, CRI-C61, CRI-R379, CRIL45, CRI-L1200, CRI-L401, CRI-P152, CRIL986, CRI-L1194, and CRI-P148. Probes pJ0205H-C and pTP5E were obtained
ET AL. from ATCC, where they have been deposited by Drs. John Wasmuth and Rosann Farber, respectively. We thank Ms. Elvi Karila and Ms. Ann-Liz Trsskelin for technical assistance and Ms. Sinikka Lindh for collecting the samples. This study has been supported by grants from the Finska Liikaresallskapet, the Sigrid Juselius Foundation, the Academy of Finland, and National Institutes of Health.
REFERENCES 1. ANDERSON, M. A., AND GUSELLA, J. (1984). Use of cyclosporin A in establishing Epstein-Barr virus-transformed human lymphoblastoid cell lines. In Vitro 20: 856-858. 2. BISHOP, D. T., AND WESTBROOK, C. (1990). Report of the committee on the genetic constitution of chromosome 5. Cytogenet. Cell Genct. 66: 111-117. 3. DONIS-KELLER, H., GREEN, P., HEI,MS, C., CARTINHOUR, S., WEIFFENBACH, B., STEPHENS, K., KEITH, T. P., BOWDEN, D. W., SMITH, D. R., LANDER, E. S., B-IN, D., AKOTS, G., RISING, M. B., PARKER, C., POWF.RS,J. A., WA=, D. E., KAUFFMAN, T. R., BRICKER, A., PHIPPS, P., MULLER-KAHLE, H., FULTON, T. R., NG, S., SCHUMM, J. W., BRAMAN, J. C., KNOWLTON, R. G., BARKER, D. F., CROOKS, S. M., LINCOLN, S. E., DALY, M. J., AND ABRAHAMSON, J. (1987). A genetic linkage map of the human genome. Cell 51: 319-337. 4. ELIMA, K., KAITILA, I., MIKONOJA, L., ELONSALO, U., PELTONEN, L., AND VUORIO, E. (1989). Exclusion of the COLPAl gene as the mutation site in diastrophic dysplasia. J. Med. Genet. 26: 314-319. 5. FARBER, R. A., PHALEN, T., NEUMAN, W. L., LE BEAU, M. M., WASMUTH, J. J., AND DOBBS, M. (1988). An anonymous DNA segment pTP5E (D5S70) maps to the long arm of chromosome 5 and identifies a TaqI polymorphism. Nucleic Acids Res. 16: 2360. 6. GIUFFRA, L. A., KENNEDY, J. L., CASTIGLIONE, C. M., EVANS, R. M., WASMIJTH, J. J., AND KIDD, K. K. (1988). Glucocorticoid receptor maps to the distal long arm of chromosome 5. Cytogenet. Cell Genet. 49: 313-314. 7. GRANDY, D. K., QUN-YONG, Z., ALL,EN, L., Lrrr, R., MAGENIS, E., CIVELLI, O., AND Lrrr, M. (1990). A human dopamine receptor gene is located on chromosome 5 at q35.1 and identifies an EcoRI RFLP. Am. J. Hum. Genet. 47: 828-834. 8. HKSTBACKA, J., KAITILA, I., SISTONFN, P., AND DE LA CHAPELLE, A. (1990). Diastrophic dysplasia gene maps to the distal long arm of chromosome 5. Proc. Natl. Acad. Sci. USA 87: 80564059. 9. HEITZ, D., JAYE, M., AND BIRNBAUM, D. (1990). EcoRI restriction polymorphism at the 5’ end of human acidic FGF gene. Nucleic Acids Res. 18: 3111. 10. HOLLENBERG, S. M., WEINBERGER, C., ONG, E. S., CERELLI, G., ORO, A., LEBO, R., THOMPSON, E. B., ROSE-, M. G., AND EVANS, R. M. (1985). Primary structure and expression of a functional human glucocorticoid receptor cDNA. Nature f’hzdon) 318: 635-641. 11. HORTON, W. A., RIMOIN, D. L., HOLLISTER, D. W., AND SILBERBERG, R. (1979). Diastrophic dwarfism: A histochemical and ultrastructural study of the endochondral growth plate. Pediutr. Res. 13: 904-909. 12. HUEBNER, K., NAGARAJAN, L., BESA, E., ANGEXT, E., LANGE, B. J., CANNIZARRO, L. A., VAN DER BERGHE, H., SANTOLI, D., FINAN, J., CROCE, C. M., AND NOWELL, P. C. (1990). Order of genes on human chromosome 5q with respect to 5q interstitial deletions. Am. J. Hum. Genet. 48: 26-36. 13. KAITILA, I. (1980). Diastrophic dysplasia. In “Population
A LINKAGE
14.
15. 16. 17.
18. 19.
20.
21. 22.
MAP SPANNING
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