Mammalian Genome 3: 39-41, 1992
9 Springer-VerlagNew York Inc. 1992
Localization of genes and anonymous DNA probes on the short arm of Chromosome 7 Klaus Wagner, Peter M. Kroisel, and Walter Rosenkranz Institute of Medical Biology and Human Genetics, Harrachgasse 21/8, A-8010 Graz, Austria Received September 9, 1991; accepted October 10, 1991
Abstract. We have previously described the cytoge-
netic analysis of two patients with Greig cephalopolysyndactyly syndrome (GCPS) and various microdeletions on the short arm of Chromosome (Chr) 7. Using genes and anonymous DNA probes from 7p we analyzed the DNA of our patients for loss of heterozygosity, or we determined the copy number by semiquantitative Southern hybridization. We have been able to show hemizygosity for the genes of INHBA, IGFBP1 and GLI3 in both patients and therefore can give the chromosomal assignment 7p12.3-p13. CRIR944 and CRI-P137 map to the same region, whereas CRI-$207 can be assigned to 7p13-p14.2; TM102L, TS93, TS194, TM77 and TN177 showed no change and these probes map distal to 7p14.2.
situ hybridization. We have been able to assign some probes to specific regions of 7p.
Materials and methods DNA was isolated by lymphoblastoid cell lines of the patients and their parents and digestion of the DNA with restriction enzymes, agarose gel electrophoresis, and Southern blotting was performed according to standard protocols (Maniatis et al. 1982). Probes were labeled by the random priming methods (Feinberg and Vogelstein 1983) and hybridized as described by Church and Gilbert (1984). The probe for INHBA was kindly provided by A. Singh, cDNA for IGFBP1 was a gift ofO. J~inne and pGLI3 HH-HaelII 640 was a gift of B. Vogelstein. TM102L, TS93, TS194, TM77, and TN177 were obtained from L.C. Tsui and CRI-P137, -R944 Ca 3.4 kb EcoRI subclone was used for hybridization as described by Drabkin and coworkers (1989) and -$207 from Collaborative Research, Inc., USA. pJ3.11 from the long arm of Chr 7 was kindly provided by J. Schmidtke and was used as internal control for quantification of
Introduction
GCPS is characterized by polysyndactyly of fingers and toes and minor cranio-facial abnormalities (Greig 1926). The disorder is inherited autosomal dominant and has been mapped to 7p13 by the analysis of translocation chromosomes in Greig families (Tommerup and Nielsen 1983; Krtiger et al. 1989) and by linkage analysis with EGFR (Brueton et al. 1988). Rosenkranz and colleagues (1989) first described two Greig patients with chromosomal visible microdeletions, and we have been able to show hemizygosity for PGAM2 and TCRG by semiquantitative Southern hybridization (Wagner et al. 1990) and loss of heterozygosity for TNI57 (D7S145; Wagner et al. 1991). For a further characterization of this chromosomal region around the Greig locus, we used several genes and anonymous DNA probes that have been mapped to the short arm of Chr 7 by somatic cell hybrids or by chromosomal in
Offprint requests to: K. Wagner
Fig. 1. DNA of the Greig patients BC (2) and IR (5) and their fathers (1 ;4) and mothers (3;6) was digested with HindIII for hybridization with INHBA; BgllI for detection of the restriction fragment length polymorphism (RFLP) for IGFBP1; and with HindlII for hybridization with a subclone of GLI3.
K. Wagner et al.: Genes deleted in Greig patients
40 Table 1. Gene copy number for Greig patients)
TMIO2L
Patient Probe
BC b
IR b
IGFBP1 INHBA GLI3
0.51 (0.06) 0.53 (0.04) 0.45 (0.07)
0.48 (0.04) 0.49 (0.06) 0.53 (0.05)
TS93 TS194 TM77 TN177
a Copy number was determined as described by McCormick and colleagues (1989). In brief, after densitometric quantification of the autoradiographic signals, the ratio (gene probe/control probe p J3.11) of the patient divided by this ratio in a normal individuum (parents) should equal 0.5 if the genomic region is deleted, whereas 1.0 indicates a normal copy number. b Mean value (-+ standard deviation; calculations have been performed dividing the value of the patient with the values of all normal persons on a single blot).
I B( IR
I
I INHBA | IGFBP1 IGLI3
$207 P137 R944
hybridization signals after stripping the blots and rehybridization with the specific probe. Copy numbers of genes or anonymous DNA probes were determined as described by McCormick and colleagues (1989).
Results and discussion
In patient BC a de novo interstitial deletion del(7) (p12.3-p14.2) has been detected by chromosomal high resolution banding, whereas in patient I R a deletion del(7) (pll.2-p13) due to a de novo translocation t(7;20) has been described previously (Wagner et al. 1990). A deletion of the genomic regions for the genes INHBA, IGFBP1 and GLI3 can be detected after densitometric evaluation of the hybridigation signals shown in Fig. 1 and the calculated gene copy numbers are summarized in,Table 1. All three probes have been deleted in both patients and the genes can be assigned to the genomic region 7p 12.3-p 13. Recently, Vortkamp and co-workers (1991) described the disruption of the GLI3 gene in three Greig families with translocation chromosomes. Therefore, this gene may have a causative role in the development of the disease. Anonymous D N A probes that have been deleted in both patients and map to the same region are CRI-R944 and CRI-P137. A more distal location can be given for the probe CRI-S207, as a loss of heterzygosity (the allele of his father is missing in patient BC) has been detected only in one patient. This finding is concordant with our pre-
Fig. 2. Localization of genes and anonymous DNA probes on the short arm of Chr 7. Note that the order of genes in the region 7p 14,2p13 is arbitrary.
vious description of the deletion of TCRG because both probes are closely physically linked and have been mapped to a common 160 kb SfiI fragment (Drabkin et al. 1989). From our data, the localization of TCRG to 7p15 (Murre et al. 1985) should be changed to 7p13-14.2. A more proximal location than 7p15 has been also suggested in a recent paper (Bensmana et al. 1991). All other anonymous DNA probes we have investigated showed either heterozygosity of the DNA of the patient or a normal copy number after calculation of the densitometric data (Table 2) and can be mapped distal to 7p14.2 taking into account our results and previous mapping data (Barker et al. 1987; Rommens et al. 1988; Lathrop et al. 1989). The breakpoints on the short arm of Chr 7 of our Greig patients and the mapping of DNA probes described in this study are delineated in Fig. 2.
Table 2. Copy number of anonymous DNA probes, a Patient
Acknowledgments. We would like to thank Andrea Renner for ex-
Probe
BC
IR
TM102L TS93 TS194 TM77 TN177 $207 P137 R944
Heterozygous 0.98 (0.05) Heterozygous Heterozygous 1.12 (0.14) Loss of heterozygosity Loss of heterozygosity 0.55 (0.06)
1.05 (0.08) Heterozygous 0.90 (0.12) 0.95 (0.08) 1.07 (0.09) 0.95 (0.07) 0.47 (0.05) 0.48 (0.04)
See Table 1.
cellent technical assistance. This work was supported by grant P7785-Med of the Fonds zur F6rderung von Wissenschaft und Forschung, Wien.
References Barker, D., Green, P., Knowlton, R., Schumm, J., Lander, E., Oliphant, A., Willard, H., Akots, G., Brown, V., Gravius, T., Helms, C., Nelson, C., Parker, C., Rediker, K., Rising, M., Watt, D., Weiffenbach, B-, and Donis-Keller, H.: Genetic linkage map
K. Wagner et al.: Genes deleted in Greig patients of human chromosome 7 with 63 DNA markers. Proc Natl Acad Sci USA 84: 8006-8010, 1987. Bensmana, M., Mattei, M.G., and Lefranc, M.P.: Localization of the human T-cell receptor 7 locus (TCRG) to 7p14-p15 by in situ hybridization. Cytogenet Cell Genet 56: 31-32, 1991. Brueton, L., Huson, S.M., Winter, R.M., and Williamson, R.: Chromosomal localisation of a developmental gene in man: Direct DNA analysis demonstrates that Greig cephalopolysyndactyly maps to 7p13. A m J M e d Genet 31: 79%804, 1988. Church, G.M. and Gilbert, W.: Genomic sequencing. Proc Natl Acad Sci USA 81: 1991-1995, 1984. Drabkin, H., Sage, M., Helms, C., Green, P., Gemmill, R., Smith, D., Erickson, P., Hart, I., Ferguson-Smith, A., Ruddle, F., and Tommerup, N.: Regional and physical mapping studies characterizing the Greig polysyndactyly 3;7 chromosome translocation, t(3;7) (p21.1;13). Genomics 4: 518-529, 1989. Feinberg, A.P. and Vogelstein, B.: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6-13, 1983. Greig, D.M.: Oxycephaly. Edinburgh M e d J 33: 189-218, 1926. Lathrop, G.M., O'Connell, P., Leppert, M., Nakamura, Y., Farrall, M., Tsui, L.C., Lalouel, J.M., and White, R.: Twenty-five loci form a continuous linkage map of markers for human chromosome 7. Genomics 5: 866-873, 1989. Kr/iger, G., Grtz, J., Kvist, U., Dunker, H., Erfurth, F., Pelz, L., and Zech, L.: Greig syndrome in a large kindred due to reciprocal chromosome translocation t(6;7) (q27;p13). A m J Med Genet 32: 411-416, 1989. Maniatis, T., Fritsch, E.F., and Sambrook, J: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1982. McCormick, M.K., Schinzel, A., Petersen, M.B., Stetten, G.,
41 Driscoll, D.J., Cantu, E.S., Tranebjaerg, L., Mikkelsen, M., Watkins, P.C., and Antonarakis, S.E.: Molecular genetic approach to the characterization of the "Down Syndrome Region" of chromosome 21. Genomics 5: 325-331, 1989. Murre, C., Waldmann, R.A., Morton, C.C., Bongiovanni, K.F., Waldmann, T.A., Shows, T.B., and Seidman, J.G.: Human ~/ chain genes are rearranged in leukaemic T cells and map to the short arm of chromosome 7. Nature 316: 549-552, 1985. Rommens, J.M., Zengerling, S., Bums, J., Melmer, G., Kerem, B.S., Plavsic, N., Zsiga, M., Kennedy, D., Markiewicz, D., Rozmahel, R., Riordan, J.R., Buchwald, M., and Tsui, L.C.: Identification and regional localization of DNA markers on chromosome 7 for the cloning of the cystic fibrosis gene. A m J Hum Genet 43: 645-663, 1988. Rosenkranz, W., Kroisel, P.M., and Wagner, K.: Deletion of the EGFR gene in one of two patients with Greig cephalopolysyndactyly syndrome and microdeletion of chromosome 7p. Cytogenet Cell Genet 51: 1069, 1989. Tommerup, N. and Nielsen, F.: A familial reciprocal translocation t(3;7) (p21;p13) associated with the Greig polysyndactylycraniofacial anomalies syndrome. A m J Med Genet 16:313-321, 1983. Vortkamp, A., Gessler, M., and Grzeschik, K.H.: GLI3 zinc-finger gene interrupted by translocations in Greig syndrome families. Nature 352: 539-540, 1991. Wagner, K., Kroisel, P.M., and Rosenkranz W.: Molecular and cytogenetic analysis in two patients with microdeletions of 7p and Greig syndrome: Hemizygosity for PGAM2 and TCRG genes. Genomics 8: 487--491, 1990. Wagner, K., Kroisel, P.M., and Rosenkranz, W.: A TaqI RFLP detected by the probe TN157 (D7S145) at the human chromosome region 7p13-14.2. Nucl Acids Res 19: 1170, 1991.
9 Springer-VerlagNew York Inc. 1992
Localization of genes and anonymous DNA probes on the short arm of Chromosome 7 Klaus Wagner, Peter M. Kroisel, and Walter Rosenkranz Institute of Medical Biology and Human Genetics, Harrachgasse 21/8, A-8010 Graz, Austria Received September 9, 1991; accepted October 10, 1991
Abstract. We have previously described the cytoge-
netic analysis of two patients with Greig cephalopolysyndactyly syndrome (GCPS) and various microdeletions on the short arm of Chromosome (Chr) 7. Using genes and anonymous DNA probes from 7p we analyzed the DNA of our patients for loss of heterozygosity, or we determined the copy number by semiquantitative Southern hybridization. We have been able to show hemizygosity for the genes of INHBA, IGFBP1 and GLI3 in both patients and therefore can give the chromosomal assignment 7p12.3-p13. CRIR944 and CRI-P137 map to the same region, whereas CRI-$207 can be assigned to 7p13-p14.2; TM102L, TS93, TS194, TM77 and TN177 showed no change and these probes map distal to 7p14.2.
situ hybridization. We have been able to assign some probes to specific regions of 7p.
Materials and methods DNA was isolated by lymphoblastoid cell lines of the patients and their parents and digestion of the DNA with restriction enzymes, agarose gel electrophoresis, and Southern blotting was performed according to standard protocols (Maniatis et al. 1982). Probes were labeled by the random priming methods (Feinberg and Vogelstein 1983) and hybridized as described by Church and Gilbert (1984). The probe for INHBA was kindly provided by A. Singh, cDNA for IGFBP1 was a gift ofO. J~inne and pGLI3 HH-HaelII 640 was a gift of B. Vogelstein. TM102L, TS93, TS194, TM77, and TN177 were obtained from L.C. Tsui and CRI-P137, -R944 Ca 3.4 kb EcoRI subclone was used for hybridization as described by Drabkin and coworkers (1989) and -$207 from Collaborative Research, Inc., USA. pJ3.11 from the long arm of Chr 7 was kindly provided by J. Schmidtke and was used as internal control for quantification of
Introduction
GCPS is characterized by polysyndactyly of fingers and toes and minor cranio-facial abnormalities (Greig 1926). The disorder is inherited autosomal dominant and has been mapped to 7p13 by the analysis of translocation chromosomes in Greig families (Tommerup and Nielsen 1983; Krtiger et al. 1989) and by linkage analysis with EGFR (Brueton et al. 1988). Rosenkranz and colleagues (1989) first described two Greig patients with chromosomal visible microdeletions, and we have been able to show hemizygosity for PGAM2 and TCRG by semiquantitative Southern hybridization (Wagner et al. 1990) and loss of heterozygosity for TNI57 (D7S145; Wagner et al. 1991). For a further characterization of this chromosomal region around the Greig locus, we used several genes and anonymous DNA probes that have been mapped to the short arm of Chr 7 by somatic cell hybrids or by chromosomal in
Offprint requests to: K. Wagner
Fig. 1. DNA of the Greig patients BC (2) and IR (5) and their fathers (1 ;4) and mothers (3;6) was digested with HindIII for hybridization with INHBA; BgllI for detection of the restriction fragment length polymorphism (RFLP) for IGFBP1; and with HindlII for hybridization with a subclone of GLI3.
K. Wagner et al.: Genes deleted in Greig patients
40 Table 1. Gene copy number for Greig patients)
TMIO2L
Patient Probe
BC b
IR b
IGFBP1 INHBA GLI3
0.51 (0.06) 0.53 (0.04) 0.45 (0.07)
0.48 (0.04) 0.49 (0.06) 0.53 (0.05)
TS93 TS194 TM77 TN177
a Copy number was determined as described by McCormick and colleagues (1989). In brief, after densitometric quantification of the autoradiographic signals, the ratio (gene probe/control probe p J3.11) of the patient divided by this ratio in a normal individuum (parents) should equal 0.5 if the genomic region is deleted, whereas 1.0 indicates a normal copy number. b Mean value (-+ standard deviation; calculations have been performed dividing the value of the patient with the values of all normal persons on a single blot).
I B( IR
I
I INHBA | IGFBP1 IGLI3
$207 P137 R944
hybridization signals after stripping the blots and rehybridization with the specific probe. Copy numbers of genes or anonymous DNA probes were determined as described by McCormick and colleagues (1989).
Results and discussion
In patient BC a de novo interstitial deletion del(7) (p12.3-p14.2) has been detected by chromosomal high resolution banding, whereas in patient I R a deletion del(7) (pll.2-p13) due to a de novo translocation t(7;20) has been described previously (Wagner et al. 1990). A deletion of the genomic regions for the genes INHBA, IGFBP1 and GLI3 can be detected after densitometric evaluation of the hybridigation signals shown in Fig. 1 and the calculated gene copy numbers are summarized in,Table 1. All three probes have been deleted in both patients and the genes can be assigned to the genomic region 7p 12.3-p 13. Recently, Vortkamp and co-workers (1991) described the disruption of the GLI3 gene in three Greig families with translocation chromosomes. Therefore, this gene may have a causative role in the development of the disease. Anonymous D N A probes that have been deleted in both patients and map to the same region are CRI-R944 and CRI-P137. A more distal location can be given for the probe CRI-S207, as a loss of heterzygosity (the allele of his father is missing in patient BC) has been detected only in one patient. This finding is concordant with our pre-
Fig. 2. Localization of genes and anonymous DNA probes on the short arm of Chr 7. Note that the order of genes in the region 7p 14,2p13 is arbitrary.
vious description of the deletion of TCRG because both probes are closely physically linked and have been mapped to a common 160 kb SfiI fragment (Drabkin et al. 1989). From our data, the localization of TCRG to 7p15 (Murre et al. 1985) should be changed to 7p13-14.2. A more proximal location than 7p15 has been also suggested in a recent paper (Bensmana et al. 1991). All other anonymous DNA probes we have investigated showed either heterozygosity of the DNA of the patient or a normal copy number after calculation of the densitometric data (Table 2) and can be mapped distal to 7p14.2 taking into account our results and previous mapping data (Barker et al. 1987; Rommens et al. 1988; Lathrop et al. 1989). The breakpoints on the short arm of Chr 7 of our Greig patients and the mapping of DNA probes described in this study are delineated in Fig. 2.
Table 2. Copy number of anonymous DNA probes, a Patient
Acknowledgments. We would like to thank Andrea Renner for ex-
Probe
BC
IR
TM102L TS93 TS194 TM77 TN177 $207 P137 R944
Heterozygous 0.98 (0.05) Heterozygous Heterozygous 1.12 (0.14) Loss of heterozygosity Loss of heterozygosity 0.55 (0.06)
1.05 (0.08) Heterozygous 0.90 (0.12) 0.95 (0.08) 1.07 (0.09) 0.95 (0.07) 0.47 (0.05) 0.48 (0.04)
See Table 1.
cellent technical assistance. This work was supported by grant P7785-Med of the Fonds zur F6rderung von Wissenschaft und Forschung, Wien.
References Barker, D., Green, P., Knowlton, R., Schumm, J., Lander, E., Oliphant, A., Willard, H., Akots, G., Brown, V., Gravius, T., Helms, C., Nelson, C., Parker, C., Rediker, K., Rising, M., Watt, D., Weiffenbach, B-, and Donis-Keller, H.: Genetic linkage map
K. Wagner et al.: Genes deleted in Greig patients of human chromosome 7 with 63 DNA markers. Proc Natl Acad Sci USA 84: 8006-8010, 1987. Bensmana, M., Mattei, M.G., and Lefranc, M.P.: Localization of the human T-cell receptor 7 locus (TCRG) to 7p14-p15 by in situ hybridization. Cytogenet Cell Genet 56: 31-32, 1991. Brueton, L., Huson, S.M., Winter, R.M., and Williamson, R.: Chromosomal localisation of a developmental gene in man: Direct DNA analysis demonstrates that Greig cephalopolysyndactyly maps to 7p13. A m J M e d Genet 31: 79%804, 1988. Church, G.M. and Gilbert, W.: Genomic sequencing. Proc Natl Acad Sci USA 81: 1991-1995, 1984. Drabkin, H., Sage, M., Helms, C., Green, P., Gemmill, R., Smith, D., Erickson, P., Hart, I., Ferguson-Smith, A., Ruddle, F., and Tommerup, N.: Regional and physical mapping studies characterizing the Greig polysyndactyly 3;7 chromosome translocation, t(3;7) (p21.1;13). Genomics 4: 518-529, 1989. Feinberg, A.P. and Vogelstein, B.: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6-13, 1983. Greig, D.M.: Oxycephaly. Edinburgh M e d J 33: 189-218, 1926. Lathrop, G.M., O'Connell, P., Leppert, M., Nakamura, Y., Farrall, M., Tsui, L.C., Lalouel, J.M., and White, R.: Twenty-five loci form a continuous linkage map of markers for human chromosome 7. Genomics 5: 866-873, 1989. Kr/iger, G., Grtz, J., Kvist, U., Dunker, H., Erfurth, F., Pelz, L., and Zech, L.: Greig syndrome in a large kindred due to reciprocal chromosome translocation t(6;7) (q27;p13). A m J Med Genet 32: 411-416, 1989. Maniatis, T., Fritsch, E.F., and Sambrook, J: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1982. McCormick, M.K., Schinzel, A., Petersen, M.B., Stetten, G.,
41 Driscoll, D.J., Cantu, E.S., Tranebjaerg, L., Mikkelsen, M., Watkins, P.C., and Antonarakis, S.E.: Molecular genetic approach to the characterization of the "Down Syndrome Region" of chromosome 21. Genomics 5: 325-331, 1989. Murre, C., Waldmann, R.A., Morton, C.C., Bongiovanni, K.F., Waldmann, T.A., Shows, T.B., and Seidman, J.G.: Human ~/ chain genes are rearranged in leukaemic T cells and map to the short arm of chromosome 7. Nature 316: 549-552, 1985. Rommens, J.M., Zengerling, S., Bums, J., Melmer, G., Kerem, B.S., Plavsic, N., Zsiga, M., Kennedy, D., Markiewicz, D., Rozmahel, R., Riordan, J.R., Buchwald, M., and Tsui, L.C.: Identification and regional localization of DNA markers on chromosome 7 for the cloning of the cystic fibrosis gene. A m J Hum Genet 43: 645-663, 1988. Rosenkranz, W., Kroisel, P.M., and Wagner, K.: Deletion of the EGFR gene in one of two patients with Greig cephalopolysyndactyly syndrome and microdeletion of chromosome 7p. Cytogenet Cell Genet 51: 1069, 1989. Tommerup, N. and Nielsen, F.: A familial reciprocal translocation t(3;7) (p21;p13) associated with the Greig polysyndactylycraniofacial anomalies syndrome. A m J Med Genet 16:313-321, 1983. Vortkamp, A., Gessler, M., and Grzeschik, K.H.: GLI3 zinc-finger gene interrupted by translocations in Greig syndrome families. Nature 352: 539-540, 1991. Wagner, K., Kroisel, P.M., and Rosenkranz W.: Molecular and cytogenetic analysis in two patients with microdeletions of 7p and Greig syndrome: Hemizygosity for PGAM2 and TCRG genes. Genomics 8: 487--491, 1990. Wagner, K., Kroisel, P.M., and Rosenkranz, W.: A TaqI RFLP detected by the probe TN157 (D7S145) at the human chromosome region 7p13-14.2. Nucl Acids Res 19: 1170, 1991.
