GENOMICS
8,487-491
(1990)
Molecular and Cytogenetic Analysis in Two Patients with Microdeletions of 7p and Greig Syndrome: Hemizygosity for PGAMZ and TCRG Genes KLAUS WAGNER, Institute
of Medical
PETER M. KROISEL,
AND WALTER
ROSENKRANZ
Biology and Human Genetics, University of Graz, Harrachgasse Received
December
29, 1989;
revised
May
27/8, A-8010 Graz, Austria
31, 1990
the EGFR gene in one of them. Clinical findings of our patients were presented (Rosenkranz et al., 1989) and a detailed description of further clinical anomalies including a follow-up study is in preparation. High-resolution banding techniques were used for cytogenetic analysis of both patients. A large part of the short arm of chromosome 7 has been associated with developmental anomalies in anterior structures. These include craniosynostosis, oculoauricular vertebral dysplasia (Goldenhar syndrome), and GCPS. The mouse developmental mutant extra toes (Xt) was described by Johnson (1967). Winter and Huson (1988) suggested that it might be an animal model of the Greig syndrome. This assumption is supported by the very similar pattern of malformations in Xt mouse heterozygotes and in humans with Greig syndrome and, on the other hand, by the homology between mouse chromosome region 13A2-A3 and human 7~15 (Searle et al., 1987). We have established B-cell lines of the patients and their parents and have performed Southern blots with dosage analysis of these genes which either have been suggested to be involved in the pathogenesis of GCPS or have been located close to this region.
Greig cephalopolysyndactyly syndrome (GCPS) is an autosomal dominant disorder that has been mapped to 7~13. We have investigated two patients with GCPS and a cytogenetically visible microdeletion of the short arm of chromosome 7 with gene probes that have been assigned close to the proposed Greig locus. Deletion breakpoints were determined from high-resolution G- and R-banded chromosomes. In patient BC with a de nouo deletion (7pl2.37~14.2) we have found a loss of the genomic region containing the T-cell receptor 7 (TCRG) gene cluster, whereas the other patient IR with a deletion (7~11.2-7~13) due to a de nouo translocation was apparently normal for this region. Gene dosage analysis revealed a loss of the phosphoglycerate mutase muscular form (PGAMB) gene locus in both patients. Hox 1.4 and interferon-& (IFNBS) showed a normal gene dosage. Our investigations revealed the following ordering and assignments of the studied genes: PGAMB and GCPS in 7~12.3-13; TCRG in the distal part of 7~13-7~14.2; Hox 1.4 and IFNBB distal to 7~14.2. Our results suggest a location of the TCRG gene more proximal than that reported previously. Furthermore, we were able to exclude the Hox 1.4 gene from involvement in the pathogenesis of GCPS. o leso Academic press, IDC.
INTRODUCTION
MATERIALS
GCPS is a rare autosomal dominant disorder with complete penetrance but somewhat variable expressivity. It is characterized by postaxial polydactyly of the fingers, preaxial and postaxial polysyndactyly of the toes, syndactyly, and minor crania-facial abnormalities (Greig, 1926). From two independent familial translocations cosegregating with Greig syndrome (Tommerup and Nielsen, 1983; Pelz et al., 1986; Kruger et al., 1989) both involving band 7~13 there is considerable evidence that the Greig gene locus itself might be affected due to the translocation event. We have first reported two patients with Greig syndrome due to chromosomal microdeletions and a deletion of
High-Resolution
AND
METHODS
Banding
Whole blood cultures of the patients and their parents were set up for 72 h at 37°C in RPM1 1640 with 20% FCS and PHA. MTX (2 pg/ml final concentration (f.c.)) was added to the culture 24 h before harvest (b.h.). At 7 or 9 h (b.h.) 10 or 100 pg/ml BrdU (f.c.) was added. Ethidium bromide (5 pg/ml, f.c.) was added 2 h before colcemid (0.2 pg/ml, f.c., b.h.). Highresolution banding was done according to a previously described alkaline Giemsa G-type replication banding technique subsequent to RBG-banding (Kroisel and Rosenkranz, 1990). Lymphoblastoid cell lines of the 487 All
Copyright 0 1990 rights of reproduction
o&3&3-7543/90 $3.00 by Academic Press, Inc. in any form reserved.
488
WAGNER,
patients and their parents were established to Neitzel (1986) with minor modifications. Probes and Southern
KROISEL,
according
Blotting
A subclone of Hox 1.4 was kindly provided by Dr. A. Ferguson-Smith and p5A6, a subclone of TCRG, was obtained from ATCC (No. 59606). PGAM2 was kindly provided by Dr. M. Cohen-Solal and IFNB2l was a gift of Dr. P. Sehgal. Probe pJ3.11 from the long arm of chromosome 7 was used as control for signal intensity and was kindly provided by Dr. J. Schmidtke. Standard protocols were used for DNA isolation from human blood and lymphoblastoid cell lines, restriction digestion, and agarose gel electrophoresis (Maniatis et al, 1982). DNA transfer to nylon membranes (Hoefer) was accomplished using a Hoefer vacuum blotting apparatus. Probes were labeled with [a-32P]dCTP to specific activities of lo*10’ cpm/pg according to Feinberg and Vogelstein (1983). Hybridizations were carried out as described by Church and Gilbert (1984). Filters were washed at a final stringency of 0.1X SSC/O.l% SDS at 65°C and exposed to Kodak XAR films for various times. Membranes were reused several times after stripping with 0.4 M NaOH, neutralization, and prehybridization. A Bio-Rad videodensitometer 620 with one-dimensional and two-dimensional software was used for quantification of autoradiographic signals. RESULTS
(A) Deletion Breakpoint Mapping Cytogenetic results with well-contrasted complementary banding patterns of the same chromosomes allows an unequivocal identification of breakpoints. In patient BC a total loss of band 7~13 is evident, whereas in patient IR the very distal part of 7~13 is not deleted. The distal breakpoint in BC is in 7~14.2 and the more proximal one is located in 7~12.3 (Fig. 1). No further rearrangement or insertion of the deleted region was detectable up to the 850-band stage according to ISCN (1985). The deletion of IR is caused by a three breaks and reunion event leading to a 7120 translocation and a loss of the region 7~11.2 to the more distal part of 7~13 (Fig. 2).
T-cell receptor y. TCRG has been mapped to the short arm of chromosome 7 to band 7~15 by in situ hybridization (Murre et al., 1985). The probe p5A6 from the central part of the TCRG cluster had half HGM-approved 6).
nomenclature
ROSENKRANZ
the intensity in BC (Fig. 3a), whereas no reduction of the hybridization signal is apparent in patient IR (Fig. 3~). From these data we can conclude that the TCRG locus is deleted in patient BC. Furthermore, we can give a more precise localization of the TCRG locus because of the different deletions of our patients. The refined assignment is 7p13-~14.2 which is more proximal as originally described. This is in good agreement with the physical mapping data of a previous study (Drabkin et al., 1989) and with the linkage of TCRG to Xt in the mouse. Phosphoglycerate mutase 2. The muscle-specific form of phosphoglycerate mutase has been mapped to chromosome 7 to the region 7pter-q32 (Edwards et al., 1989). Fine mapping to 7p12-~13 has been reported by Mattei et al. (1989). Hybridization signals are shown for BC (Fig. 3b) and IR (Fig. 3d). As can be seen in Table 1 the signal intensity of PGAM2 is reduced in both patients. However, we cannot determine whether the gene lies proximal or distal to GCPS on the basis of our experimental data. Hox 1.4. Mapping of Hox 1.4 to 7p13-p15 has been described using in situ hybridization and mouse x human somatic cell hybrids (Ferguson-Smith et al., 1989). When we hybridized a subclone of Hox 1.4 to DNA of our two patients no reduction in the signal intensity was detectable and therefore no deletion of the corresponding genomic region had occurred (Table 1). Taking into account our high-resolution banding data we can place the Hox 1.4 gene cluster distal to 7~14.2 Interferon-&. IFNB2 has been mapped to the region 7p15-p21 using mouse X human somatic cell hybrids and chromosomal in situ hybridization (Ferguson-Smith et al., 1988). Investigation of our two patients with a cDNA clone for IFNBB (Sehgal et al., 1986) did not reveal an abnormal band localization or intensity on four different genomic Southern blots and therefore this probe resides outside the deleted genomic region (Table 1). Our data suggest the following locations and order of genes on the short arm of chromosome 7 (Fig. 4): PGAMB and GCPS, 7p12.3-~13; TCRG, 7p13-~14.2; IFNB2 and Hox 1.4, distal to 7~14.2. DISCUSSION
(B) Gene Dosage Analysis
1 The terleukin
AND
for IFNBZ
is now IL6
(in-
Cytogenetic studies revealed a de novo deletion of the short arm of chromosome 7 in two patients with GCPS. Our staining technique, which allows sequential R- and G-banding of high quality of the same metaphase chromosomes, is undoubtably helpful in refined breakpoint determination. Because the deleted regions of BC and IR show overlaps, GCPS can be assigned to band 7p12.3-~13. A few familial trans-
GENE
a
DELETIONS
IN
GREIG
489
SYNDROME
b
FIG. 1. Partial karyotype of chromosomes 7 of patient b) of RBG(left) and G-type replication (rigbt) banding pair and the corresponding ISCN scheme at the SO-band
C BC with a de nova interstitial deletion de1 (7) (p12.3-~14.2). Two examples (a and of the same chromosomes with the normal chromosome 7 on the left side in each stage (c).
locations involving band 7~13 without Greig syndrome have been reported (Gabarron et d., 1988). It is noteworthy that one of the breakpoints of chromosomes 7 in our patients could be located within the
a FIG. 2. Partial karyotype of chromosomes 7 and 20 of patient (p11.2-~13) due to this translocation. RBG(left) and subsequent the corresponding ISCN scheme at the 850-band stage (b).
GCPS gene. This is the more distal breakpoint in band 7~13 in IR. In contrast to this, we can predict a deletion of the whole Greig locus in patient BC. Aninteresting finding is that three of the four
b IR with a o!e nova translocation t(7;20) and a G-type replication (right) of the same metaphase
de novo deletion chromosomes
del(7) (a) and
490
WAGNER, F
BC
M
F
KROISEL,
IR
AND
ROSENKRANZ
M
J3.11 TCRG
a J3.11
w
PGAM-2
w
b
IRI
PGAM2
cl
FIG. 3. Southern blots of genomic DNA digested with Hind111 (a), BglII (b), XbaI (c), andBglI1 (d). Blots show patient BC (a and b) and patient IR (c and d), lanes M and F refer to mother and father of the patient. The upper bands represent hybridization signals of probe pJ3.11 as control for the amount of DNA loaded. The lower bands are from probe TCRG (a and c) and PGAMZ (b and d).
breakpoints on chromosome 7 in our patients are identical at the cytogenetic level with fragile sites: at band 7~11.2 of folic acid type and at 7~13 and 7~14.2 of aphidicoline type. In an investigation of the translocation chromosome t(3; 7) (~21.1; 13) TCRG shared a common SfiI fragment of 160 kb with CR1 S207, which in turn is genetically linked with 2 CM to the probes flanking the translocation breakpoint (Drabkin et al, 1989). Patient BC has lost at least a part of the TCRG gene cluster. The relevance of this deletion to his immunological development has yet to be established. Hox 1.4 is an important gene in the regulation of embryonic development. A possible connection between the Hox 1 cluster and the GCPS gene has been suggested (Ferguson-Smith et al., 1989). From our results we can assign this gene locus distal to TCRG and, taking into account the deletion breakpoints, we can exclude the Hox 1 region from being involved in GCPS. The local-
TABLE Gene Copy Number
1 of Patients” Patient
Gene IFNBP Hox 1.4 TCRG PGAMS
BC” 1.04 0.99 0.53 0.41
(0.08) (0.12) (0.04) (0.08)
IR* 1.03 1.04 1.06 0.50
FIG. 4. Schematic representation of the deletions tients BC and IR (left) and corresponding breakpoints cated by arrows as well as gene locations (right).
ization of IFNBB distal to the deletions of our patients is in good agreement with previous mapping to 7~15~21. Recently Peters et al. (1989) reported localization of biliverdin reductase (BLVR) to mouse chromosome 2. Interestingly, this locus is in close linkage to genes involved in limb and crania-facial dysmorphology in the mouse. The authors discussed a cluster of genes for crania-facial and limb development in the common ancestor of man and mouse that remained intact in the region 7p between BLVR and TCRG in man. On the other hand, synteny was broken up during evolution in the mouse and loci from the short arm of chromosome 7 can be found on four different mouse chromosomes (Searle et aZ., 1987). Our study demonstrates that a combination of high-resolution banding with molecular analysis in patients with chromosomal microdeletions provides a valuable approach for improved gene localization.
(0.14) (0.08) (0.12) (0.09)
a Gene copy number was determined as described (15). In brief, after densitometric quantification of the autoradiographic signals the ratio (gene probe/control probe pJ3.11) of the patient divided by this ratio in a normal individuum (parents) should equal 0.5 if the genomic region is deleted or 1.0 for a normal gene copy number. * Mean value (standard deviation).
in both paare indi-
ACKNOWLEDGMENTS This work was supported Fiirderung von Wissenschaft
in part by grants of the und Forschung, Wien.
Fonds
sur
REFERENCES 1.
CHURCH, G. M., AND GILBERT, W. (1984). Genomic ing. Proc. Natl. Acad. Sci. USA 81: 199-1995.
sequenc-
GENE 2.
DELETIONS
IN
H., SAGE, M., HELMS, C., GREEN, P., GEMMILL, R., D., ERICKSON, P., HART, I., FERGUSON-SMITH, A., RUDDLE, F., AND TOMMERUP, N. (1989). Regional and physical mapping studies characterizing the Greig polysyndactyly 3;7 chromosome translocation, t(3;7)(p21.1; 13). Genomics 4: 518-529. EDWARDS, Y. H., SAKODA, S., SCHON, E., AND POVEY, S. (1989). The gene for human muscle-specific phosphoglycerate mutase, PGAMZ, mapped to chromosome 7 by polymerase chain reaction. Genomics 5: 948-951.
GREIG
D-KIN,
lecular Cloning: A Laboratory Manual,” Laboratory, Cold Spring Harbor, NY.
SMITH,
3.
4.
FEINBERG, A. P., AND VOGELSTEIN, B. (1983). A technique radiolabeling DNA restriction endonuclease fragments high specific activity. Anal. Biochem. 132: 6-13.
5.
FERGUSON-SMITH, A. C., CHEN, Y. F., NEWMAN, M. S., MAY, L. D., SEHGAL, P. B., AND RUDDLE, F. H. (1988). Regional localization of the interferon-&/B-cell stimulatory factor 2/ hepatocyte stimulating factor gene to human chromosome 7p15-~21. Gerwmics 2: 203-208.
6.
FERGUSON-SMITH, A., FIENBERG, A., AND RUDDLE, F. (1989). Isolation, chromosomal localization, and nucleotide sequence of the human HOX 1.4 homeobox. Genomics 6: 250-258. GABARRON, J., GLOVER, G., JIMENEZ, A., SALAS, P., PEREZBRYAN, J., AND PARRA, M. J. (1988). Chromosomal imbalance in the offspring of translocation carriers involving 7p: Further contribution with three cases to the partial trisomy 7p phenotype. Clin. Genet. 33: 211-219. GREIG, D. M. (1926). Oxycephaly. Edinburgh Med. J. 33: 189-218. ISCN (1985). “An International System for Human Cytogenetic Nomenclature” (D. G. Harnden and H. P. Klinger, Eds.), published in collaboration with Cytogenet. Cell Genet., Karger, Basel. JOHNSON, D. R. (1967). Extra-toes: A new mutant gene causing multiple abnormalities in the mouse. J. Embryol. Exp. Morphol. 17: 543-581.
I.
8. 9.
10.
11.
12.
13.
MANIATIS,
T., FRITSCH,
E. F., AND SAMBROOK,
J. (1982).
“Mo-
Cold
Spring
Harbor
14.
MAYI+EI, M. G., CASTELLA-ESCOLA, J., OJICIUS, D., PASSAGE, E., VALENTIN, C., AND COHEN-SOLAL, M. (1989). In situ mapping of phosphoglycerate mutase muscular form to the human chromosome 7. Cytogenet. Cell Genet. 51: 1041.
15.
MCCORMICK, M. K., SCHINZEL, A., PETERSEN, M. B., STETTEN, G., DRISCOLL, D. J., CANTU, E. S., TUNEBJAERG, L., MIKKELSEN, M., WATKINS, P. C., AND ANTONARAKIS, S. E. (1989). Molecular genetic approach to the characterization of the “Down syndrome region” of chromosome 21. Genomics 6: 325-331.
16.
MURRE, C., WALDMANN, R. A., MORTON, C. C., BONGIOVANNI, K. F., WALDMANN, T. A., SHOWS, T. B., ANLI SElDMAN, J. G. (1985). Human gamma chain genes are rearranged in leukaemic T cells and map to the short arm of chromosome 7. Nature flkdon) 316: 549-552.
17.
NEITZEL, H. (1986). A routine method of permanent growing lymphoblastoid 73: 320-326.
18.
PELZ, L., KRUEGER, G., AND G&Z, L. (1986). The Greig cephalopolysyndactyly syndrome. Helv. Paediatr. Acta 41: 381. PETERS, J., BALL, S. T., AND VON DEXMLING, A. (1989). LocaIization of BLVR, biliverdin reductase, on mouse chromosome 2. Genomics 6: 270-274.
for to
KROISEL, P. M., AND ROSENKRANZ, W. (1990). High resolution banding of an unusual translocation in recurrent abortions. Clin. Genet. 37: 230-234. KR~~GER, G., GBTz, J., KVIST, U., DUNKER, H., ERFURTH, F., PELZ, L., AND ZECH, L. (1989). Greig syndrome in a large kindred due to reciprocal chromosome translocation t(6;7)(q27;p13). Amer. J. Med. Genet. 32: 411-416.
491
SYNDROME
19.
for the establishment cell lines. Hum. Genet.
20.
ROSENKRANZ, W., KROISEL, P. M., AM) WAGNER, Deletion of the EGFR gene in one of two patients cephalopolysyndactyly syndrome and microdeletion mosome 7p. Cytogenet. Cell Genet. 51: 1069.
21.
SEHGAL, P. B., ZILBERSTEIN, A., RUGGIERI, R. M., MAY, L. T., FERGUSON-SMITH, A., SLATE, D. L., REVEL, M., AND RUDDLE, F. H. (1986). Human chromosome 7 carries the 8, interferon gene. Proc. Natl. Acad. Sci. USA 83: 5219-5222. SEARLE, A. G., PETERS, J., LYON, M. F., EVANS, E. P., EDWARDS, J. H., AND BUCKLE, V. J. (1987). Chromosome maps of man and mouse, III. Genomics 1: 3-18. TOMMERUP, N., AND NIELSEN, F. (1983). A familial reciprocal translocation t(3;7)(p2Ll;p13) associated with the Greig polysyndactyly-craniofacial anomalies syndrome. Amer. J. Med. Genet. 16: 313-321. WINTER, R. M., AND HUSON, S. M. (1988). Greig cephalopolysyndactyly syndrome: A possible mouse homologue (Xt-extra toes). Amer. J. Med. Genet. 31: 793-798.
22.
23.
24.
K. (1989). with Greig of chro-
8,487-491
(1990)
Molecular and Cytogenetic Analysis in Two Patients with Microdeletions of 7p and Greig Syndrome: Hemizygosity for PGAMZ and TCRG Genes KLAUS WAGNER, Institute
of Medical
PETER M. KROISEL,
AND WALTER
ROSENKRANZ
Biology and Human Genetics, University of Graz, Harrachgasse Received
December
29, 1989;
revised
May
27/8, A-8010 Graz, Austria
31, 1990
the EGFR gene in one of them. Clinical findings of our patients were presented (Rosenkranz et al., 1989) and a detailed description of further clinical anomalies including a follow-up study is in preparation. High-resolution banding techniques were used for cytogenetic analysis of both patients. A large part of the short arm of chromosome 7 has been associated with developmental anomalies in anterior structures. These include craniosynostosis, oculoauricular vertebral dysplasia (Goldenhar syndrome), and GCPS. The mouse developmental mutant extra toes (Xt) was described by Johnson (1967). Winter and Huson (1988) suggested that it might be an animal model of the Greig syndrome. This assumption is supported by the very similar pattern of malformations in Xt mouse heterozygotes and in humans with Greig syndrome and, on the other hand, by the homology between mouse chromosome region 13A2-A3 and human 7~15 (Searle et al., 1987). We have established B-cell lines of the patients and their parents and have performed Southern blots with dosage analysis of these genes which either have been suggested to be involved in the pathogenesis of GCPS or have been located close to this region.
Greig cephalopolysyndactyly syndrome (GCPS) is an autosomal dominant disorder that has been mapped to 7~13. We have investigated two patients with GCPS and a cytogenetically visible microdeletion of the short arm of chromosome 7 with gene probes that have been assigned close to the proposed Greig locus. Deletion breakpoints were determined from high-resolution G- and R-banded chromosomes. In patient BC with a de nouo deletion (7pl2.37~14.2) we have found a loss of the genomic region containing the T-cell receptor 7 (TCRG) gene cluster, whereas the other patient IR with a deletion (7~11.2-7~13) due to a de nouo translocation was apparently normal for this region. Gene dosage analysis revealed a loss of the phosphoglycerate mutase muscular form (PGAMB) gene locus in both patients. Hox 1.4 and interferon-& (IFNBS) showed a normal gene dosage. Our investigations revealed the following ordering and assignments of the studied genes: PGAMB and GCPS in 7~12.3-13; TCRG in the distal part of 7~13-7~14.2; Hox 1.4 and IFNBB distal to 7~14.2. Our results suggest a location of the TCRG gene more proximal than that reported previously. Furthermore, we were able to exclude the Hox 1.4 gene from involvement in the pathogenesis of GCPS. o leso Academic press, IDC.
INTRODUCTION
MATERIALS
GCPS is a rare autosomal dominant disorder with complete penetrance but somewhat variable expressivity. It is characterized by postaxial polydactyly of the fingers, preaxial and postaxial polysyndactyly of the toes, syndactyly, and minor crania-facial abnormalities (Greig, 1926). From two independent familial translocations cosegregating with Greig syndrome (Tommerup and Nielsen, 1983; Pelz et al., 1986; Kruger et al., 1989) both involving band 7~13 there is considerable evidence that the Greig gene locus itself might be affected due to the translocation event. We have first reported two patients with Greig syndrome due to chromosomal microdeletions and a deletion of
High-Resolution
AND
METHODS
Banding
Whole blood cultures of the patients and their parents were set up for 72 h at 37°C in RPM1 1640 with 20% FCS and PHA. MTX (2 pg/ml final concentration (f.c.)) was added to the culture 24 h before harvest (b.h.). At 7 or 9 h (b.h.) 10 or 100 pg/ml BrdU (f.c.) was added. Ethidium bromide (5 pg/ml, f.c.) was added 2 h before colcemid (0.2 pg/ml, f.c., b.h.). Highresolution banding was done according to a previously described alkaline Giemsa G-type replication banding technique subsequent to RBG-banding (Kroisel and Rosenkranz, 1990). Lymphoblastoid cell lines of the 487 All
Copyright 0 1990 rights of reproduction
o&3&3-7543/90 $3.00 by Academic Press, Inc. in any form reserved.
488
WAGNER,
patients and their parents were established to Neitzel (1986) with minor modifications. Probes and Southern
KROISEL,
according
Blotting
A subclone of Hox 1.4 was kindly provided by Dr. A. Ferguson-Smith and p5A6, a subclone of TCRG, was obtained from ATCC (No. 59606). PGAM2 was kindly provided by Dr. M. Cohen-Solal and IFNB2l was a gift of Dr. P. Sehgal. Probe pJ3.11 from the long arm of chromosome 7 was used as control for signal intensity and was kindly provided by Dr. J. Schmidtke. Standard protocols were used for DNA isolation from human blood and lymphoblastoid cell lines, restriction digestion, and agarose gel electrophoresis (Maniatis et al, 1982). DNA transfer to nylon membranes (Hoefer) was accomplished using a Hoefer vacuum blotting apparatus. Probes were labeled with [a-32P]dCTP to specific activities of lo*10’ cpm/pg according to Feinberg and Vogelstein (1983). Hybridizations were carried out as described by Church and Gilbert (1984). Filters were washed at a final stringency of 0.1X SSC/O.l% SDS at 65°C and exposed to Kodak XAR films for various times. Membranes were reused several times after stripping with 0.4 M NaOH, neutralization, and prehybridization. A Bio-Rad videodensitometer 620 with one-dimensional and two-dimensional software was used for quantification of autoradiographic signals. RESULTS
(A) Deletion Breakpoint Mapping Cytogenetic results with well-contrasted complementary banding patterns of the same chromosomes allows an unequivocal identification of breakpoints. In patient BC a total loss of band 7~13 is evident, whereas in patient IR the very distal part of 7~13 is not deleted. The distal breakpoint in BC is in 7~14.2 and the more proximal one is located in 7~12.3 (Fig. 1). No further rearrangement or insertion of the deleted region was detectable up to the 850-band stage according to ISCN (1985). The deletion of IR is caused by a three breaks and reunion event leading to a 7120 translocation and a loss of the region 7~11.2 to the more distal part of 7~13 (Fig. 2).
T-cell receptor y. TCRG has been mapped to the short arm of chromosome 7 to band 7~15 by in situ hybridization (Murre et al., 1985). The probe p5A6 from the central part of the TCRG cluster had half HGM-approved 6).
nomenclature
ROSENKRANZ
the intensity in BC (Fig. 3a), whereas no reduction of the hybridization signal is apparent in patient IR (Fig. 3~). From these data we can conclude that the TCRG locus is deleted in patient BC. Furthermore, we can give a more precise localization of the TCRG locus because of the different deletions of our patients. The refined assignment is 7p13-~14.2 which is more proximal as originally described. This is in good agreement with the physical mapping data of a previous study (Drabkin et al., 1989) and with the linkage of TCRG to Xt in the mouse. Phosphoglycerate mutase 2. The muscle-specific form of phosphoglycerate mutase has been mapped to chromosome 7 to the region 7pter-q32 (Edwards et al., 1989). Fine mapping to 7p12-~13 has been reported by Mattei et al. (1989). Hybridization signals are shown for BC (Fig. 3b) and IR (Fig. 3d). As can be seen in Table 1 the signal intensity of PGAM2 is reduced in both patients. However, we cannot determine whether the gene lies proximal or distal to GCPS on the basis of our experimental data. Hox 1.4. Mapping of Hox 1.4 to 7p13-p15 has been described using in situ hybridization and mouse x human somatic cell hybrids (Ferguson-Smith et al., 1989). When we hybridized a subclone of Hox 1.4 to DNA of our two patients no reduction in the signal intensity was detectable and therefore no deletion of the corresponding genomic region had occurred (Table 1). Taking into account our high-resolution banding data we can place the Hox 1.4 gene cluster distal to 7~14.2 Interferon-&. IFNB2 has been mapped to the region 7p15-p21 using mouse X human somatic cell hybrids and chromosomal in situ hybridization (Ferguson-Smith et al., 1988). Investigation of our two patients with a cDNA clone for IFNBB (Sehgal et al., 1986) did not reveal an abnormal band localization or intensity on four different genomic Southern blots and therefore this probe resides outside the deleted genomic region (Table 1). Our data suggest the following locations and order of genes on the short arm of chromosome 7 (Fig. 4): PGAMB and GCPS, 7p12.3-~13; TCRG, 7p13-~14.2; IFNB2 and Hox 1.4, distal to 7~14.2. DISCUSSION
(B) Gene Dosage Analysis
1 The terleukin
AND
for IFNBZ
is now IL6
(in-
Cytogenetic studies revealed a de novo deletion of the short arm of chromosome 7 in two patients with GCPS. Our staining technique, which allows sequential R- and G-banding of high quality of the same metaphase chromosomes, is undoubtably helpful in refined breakpoint determination. Because the deleted regions of BC and IR show overlaps, GCPS can be assigned to band 7p12.3-~13. A few familial trans-
GENE
a
DELETIONS
IN
GREIG
489
SYNDROME
b
FIG. 1. Partial karyotype of chromosomes 7 of patient b) of RBG(left) and G-type replication (rigbt) banding pair and the corresponding ISCN scheme at the SO-band
C BC with a de nova interstitial deletion de1 (7) (p12.3-~14.2). Two examples (a and of the same chromosomes with the normal chromosome 7 on the left side in each stage (c).
locations involving band 7~13 without Greig syndrome have been reported (Gabarron et d., 1988). It is noteworthy that one of the breakpoints of chromosomes 7 in our patients could be located within the
a FIG. 2. Partial karyotype of chromosomes 7 and 20 of patient (p11.2-~13) due to this translocation. RBG(left) and subsequent the corresponding ISCN scheme at the 850-band stage (b).
GCPS gene. This is the more distal breakpoint in band 7~13 in IR. In contrast to this, we can predict a deletion of the whole Greig locus in patient BC. Aninteresting finding is that three of the four
b IR with a o!e nova translocation t(7;20) and a G-type replication (right) of the same metaphase
de novo deletion chromosomes
del(7) (a) and
490
WAGNER, F
BC
M
F
KROISEL,
IR
AND
ROSENKRANZ
M
J3.11 TCRG
a J3.11
w
PGAM-2
w
b
IRI
PGAM2
cl
FIG. 3. Southern blots of genomic DNA digested with Hind111 (a), BglII (b), XbaI (c), andBglI1 (d). Blots show patient BC (a and b) and patient IR (c and d), lanes M and F refer to mother and father of the patient. The upper bands represent hybridization signals of probe pJ3.11 as control for the amount of DNA loaded. The lower bands are from probe TCRG (a and c) and PGAMZ (b and d).
breakpoints on chromosome 7 in our patients are identical at the cytogenetic level with fragile sites: at band 7~11.2 of folic acid type and at 7~13 and 7~14.2 of aphidicoline type. In an investigation of the translocation chromosome t(3; 7) (~21.1; 13) TCRG shared a common SfiI fragment of 160 kb with CR1 S207, which in turn is genetically linked with 2 CM to the probes flanking the translocation breakpoint (Drabkin et al, 1989). Patient BC has lost at least a part of the TCRG gene cluster. The relevance of this deletion to his immunological development has yet to be established. Hox 1.4 is an important gene in the regulation of embryonic development. A possible connection between the Hox 1 cluster and the GCPS gene has been suggested (Ferguson-Smith et al., 1989). From our results we can assign this gene locus distal to TCRG and, taking into account the deletion breakpoints, we can exclude the Hox 1 region from being involved in GCPS. The local-
TABLE Gene Copy Number
1 of Patients” Patient
Gene IFNBP Hox 1.4 TCRG PGAMS
BC” 1.04 0.99 0.53 0.41
(0.08) (0.12) (0.04) (0.08)
IR* 1.03 1.04 1.06 0.50
FIG. 4. Schematic representation of the deletions tients BC and IR (left) and corresponding breakpoints cated by arrows as well as gene locations (right).
ization of IFNBB distal to the deletions of our patients is in good agreement with previous mapping to 7~15~21. Recently Peters et al. (1989) reported localization of biliverdin reductase (BLVR) to mouse chromosome 2. Interestingly, this locus is in close linkage to genes involved in limb and crania-facial dysmorphology in the mouse. The authors discussed a cluster of genes for crania-facial and limb development in the common ancestor of man and mouse that remained intact in the region 7p between BLVR and TCRG in man. On the other hand, synteny was broken up during evolution in the mouse and loci from the short arm of chromosome 7 can be found on four different mouse chromosomes (Searle et aZ., 1987). Our study demonstrates that a combination of high-resolution banding with molecular analysis in patients with chromosomal microdeletions provides a valuable approach for improved gene localization.
(0.14) (0.08) (0.12) (0.09)
a Gene copy number was determined as described (15). In brief, after densitometric quantification of the autoradiographic signals the ratio (gene probe/control probe pJ3.11) of the patient divided by this ratio in a normal individuum (parents) should equal 0.5 if the genomic region is deleted or 1.0 for a normal gene copy number. * Mean value (standard deviation).
in both paare indi-
ACKNOWLEDGMENTS This work was supported Fiirderung von Wissenschaft
in part by grants of the und Forschung, Wien.
Fonds
sur
REFERENCES 1.
CHURCH, G. M., AND GILBERT, W. (1984). Genomic ing. Proc. Natl. Acad. Sci. USA 81: 199-1995.
sequenc-
GENE 2.
DELETIONS
IN
H., SAGE, M., HELMS, C., GREEN, P., GEMMILL, R., D., ERICKSON, P., HART, I., FERGUSON-SMITH, A., RUDDLE, F., AND TOMMERUP, N. (1989). Regional and physical mapping studies characterizing the Greig polysyndactyly 3;7 chromosome translocation, t(3;7)(p21.1; 13). Genomics 4: 518-529. EDWARDS, Y. H., SAKODA, S., SCHON, E., AND POVEY, S. (1989). The gene for human muscle-specific phosphoglycerate mutase, PGAMZ, mapped to chromosome 7 by polymerase chain reaction. Genomics 5: 948-951.
GREIG
D-KIN,
lecular Cloning: A Laboratory Manual,” Laboratory, Cold Spring Harbor, NY.
SMITH,
3.
4.
FEINBERG, A. P., AND VOGELSTEIN, B. (1983). A technique radiolabeling DNA restriction endonuclease fragments high specific activity. Anal. Biochem. 132: 6-13.
5.
FERGUSON-SMITH, A. C., CHEN, Y. F., NEWMAN, M. S., MAY, L. D., SEHGAL, P. B., AND RUDDLE, F. H. (1988). Regional localization of the interferon-&/B-cell stimulatory factor 2/ hepatocyte stimulating factor gene to human chromosome 7p15-~21. Gerwmics 2: 203-208.
6.
FERGUSON-SMITH, A., FIENBERG, A., AND RUDDLE, F. (1989). Isolation, chromosomal localization, and nucleotide sequence of the human HOX 1.4 homeobox. Genomics 6: 250-258. GABARRON, J., GLOVER, G., JIMENEZ, A., SALAS, P., PEREZBRYAN, J., AND PARRA, M. J. (1988). Chromosomal imbalance in the offspring of translocation carriers involving 7p: Further contribution with three cases to the partial trisomy 7p phenotype. Clin. Genet. 33: 211-219. GREIG, D. M. (1926). Oxycephaly. Edinburgh Med. J. 33: 189-218. ISCN (1985). “An International System for Human Cytogenetic Nomenclature” (D. G. Harnden and H. P. Klinger, Eds.), published in collaboration with Cytogenet. Cell Genet., Karger, Basel. JOHNSON, D. R. (1967). Extra-toes: A new mutant gene causing multiple abnormalities in the mouse. J. Embryol. Exp. Morphol. 17: 543-581.
I.
8. 9.
10.
11.
12.
13.
MANIATIS,
T., FRITSCH,
E. F., AND SAMBROOK,
J. (1982).
“Mo-
Cold
Spring
Harbor
14.
MAYI+EI, M. G., CASTELLA-ESCOLA, J., OJICIUS, D., PASSAGE, E., VALENTIN, C., AND COHEN-SOLAL, M. (1989). In situ mapping of phosphoglycerate mutase muscular form to the human chromosome 7. Cytogenet. Cell Genet. 51: 1041.
15.
MCCORMICK, M. K., SCHINZEL, A., PETERSEN, M. B., STETTEN, G., DRISCOLL, D. J., CANTU, E. S., TUNEBJAERG, L., MIKKELSEN, M., WATKINS, P. C., AND ANTONARAKIS, S. E. (1989). Molecular genetic approach to the characterization of the “Down syndrome region” of chromosome 21. Genomics 6: 325-331.
16.
MURRE, C., WALDMANN, R. A., MORTON, C. C., BONGIOVANNI, K. F., WALDMANN, T. A., SHOWS, T. B., ANLI SElDMAN, J. G. (1985). Human gamma chain genes are rearranged in leukaemic T cells and map to the short arm of chromosome 7. Nature flkdon) 316: 549-552.
17.
NEITZEL, H. (1986). A routine method of permanent growing lymphoblastoid 73: 320-326.
18.
PELZ, L., KRUEGER, G., AND G&Z, L. (1986). The Greig cephalopolysyndactyly syndrome. Helv. Paediatr. Acta 41: 381. PETERS, J., BALL, S. T., AND VON DEXMLING, A. (1989). LocaIization of BLVR, biliverdin reductase, on mouse chromosome 2. Genomics 6: 270-274.
for to
KROISEL, P. M., AND ROSENKRANZ, W. (1990). High resolution banding of an unusual translocation in recurrent abortions. Clin. Genet. 37: 230-234. KR~~GER, G., GBTz, J., KVIST, U., DUNKER, H., ERFURTH, F., PELZ, L., AND ZECH, L. (1989). Greig syndrome in a large kindred due to reciprocal chromosome translocation t(6;7)(q27;p13). Amer. J. Med. Genet. 32: 411-416.
491
SYNDROME
19.
for the establishment cell lines. Hum. Genet.
20.
ROSENKRANZ, W., KROISEL, P. M., AM) WAGNER, Deletion of the EGFR gene in one of two patients cephalopolysyndactyly syndrome and microdeletion mosome 7p. Cytogenet. Cell Genet. 51: 1069.
21.
SEHGAL, P. B., ZILBERSTEIN, A., RUGGIERI, R. M., MAY, L. T., FERGUSON-SMITH, A., SLATE, D. L., REVEL, M., AND RUDDLE, F. H. (1986). Human chromosome 7 carries the 8, interferon gene. Proc. Natl. Acad. Sci. USA 83: 5219-5222. SEARLE, A. G., PETERS, J., LYON, M. F., EVANS, E. P., EDWARDS, J. H., AND BUCKLE, V. J. (1987). Chromosome maps of man and mouse, III. Genomics 1: 3-18. TOMMERUP, N., AND NIELSEN, F. (1983). A familial reciprocal translocation t(3;7)(p2Ll;p13) associated with the Greig polysyndactyly-craniofacial anomalies syndrome. Amer. J. Med. Genet. 16: 313-321. WINTER, R. M., AND HUSON, S. M. (1988). Greig cephalopolysyndactyly syndrome: A possible mouse homologue (Xt-extra toes). Amer. J. Med. Genet. 31: 793-798.
22.
23.
24.
K. (1989). with Greig of chro-
