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Localization of the Miller-Dieker critical region is proximal to locus D17S34 (p144D6) in 17p13.3
GENOMICS
7,621~624
(1990)
SHORT COMMUNICATION Localization of the Miller-Dieker Critical Region Is Proximal to Locus D17534 (pl44D6) in 17~13.3 CHARLES E. ScHwARTz,*,tpl SUZANNE M. SAUER,*-* ANGELA M. BROWN,* JAMES E. DIVELBISS,$ AND SHIVANAND R. PATIL* *Greenwood
Genetic Center, Greenwood, South Carolina, tDepartment of Medical Genetics, Self Memorial Greenwood, South Carolina, and *Department of Pediatrics, Division of Medical Genetics, University of Iowa Hospitals and Clinics, Iowa City, Iowa Received
November
10, 1989;
revised
March
Hospital,
21, 1990
(D17S34), was deleted in some, but not all, of these MDS patients. Schwartz et al. (1988) suggested that this finding could mean locus D17S34 either defined the distal limit of the MDS critical region or was a portion of the critical region not deleted in some of the patients. These patients, therefore, exhibited MDS because of the absence of the region containing D17S.5 and D17S28. We have studied a male patient with a satellited short arm of chromosome 17 (Fig. 1). There was no apparent absence of satellites on any of the acrocentric chromosomes (13, 14, 15, 21,22), excluding the possibility of a reciprocal translocation. This abnormality was detected prenatally by amniocentesis due to advanced
A child with normal growth and development and the abnormal karyotype 46,XY,17ps, was analyzed using molecular probes localized to 17~ 13. The results indicated the presence of two copies of the probes YNZ22.1 (Dl755) and YNH37.3 (D17528), previously shown to be deleted in all Miller-Dieker (MDS) patients studied. However, the patient was hemizygous for probe p144D6 (D17534), which is absent in approximately 75% of the MDS patients. As the patient is active at 9 months of age, with no clinical signs of MDS, the results conArm that the absence of locus D17S34 does not lead to the phenotypic expression of MDS. Furthermore, this deletion should assist in defining tbe distal limits of this contiguous gene syndrome. 8 lssoAcademic PWE,IIIC.
The Miller-Dieker syndrome (MDS) is an example of the group of disorders known as “contiguous gene syndromes” (Schmickel, 1986). MDS exhibits a combination of recognizable facial malformations, retarded growth, and lissencephaly (Dobyns et al., 1984). It is quite often associated with a deletion of chromosome band 17~13 (Dobyns et al., 1983; Stratton et al., 1984). Recently, Schwartz et al. (1988) and vanTunien et al. (1988) demonstrated that although all cases of MDS may not exhibit a visible deletion of 17p, all apparently have a submicroscopic deletion. Both of these groups found two highly polymorphic DNA probes, YNZ22.1 (D17S5) and YNH37.3 (D17S28), to be deleted in all of their MDS patients. A third probe, p144D6 ‘To whom reprint requests should be addressed at Greenwood Genetic Center, 1 Gregor Mendel Circle, Greenwood, SC 29646. * Present address: Department of Medical Genetics, Indiana University Medical Center, Indianapolis, IN.
FIG. normal 621
1. Partial chromosome
karyotypes of the patient from two cells showing 17 and satellited chromosome (arrow). osss-7543/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
622
SHORT
A.
COMMUNICATION
C.
8.
FIG. 2. Restriction fragments detected by the three chromosome 17 probes digestion; (B) p144D6 after ZizqI digestion; (C) YNH37.3 after MspI digestion. labeling of probes were done as previously described (8). Filters were sequentially p144D6 (MspI).
maternal age. Both parents were found to be chromosomally normal. On examination at 9 months of age, the patient was found to be active with good verbal and motor skills and normal growth. Except for epi-
after digestion of genomic DNA. F, father; Pt, patient; M, mother. probed with YNZ22.1 and p144D6
(A) YNZ22.1 after Southern transfers (TaqI) or YNH37.3
‘Z’oqI and and
canthal folds and broad feet, his physical examination was normal. Given the lack of any clinical signs of MDS in the patient, molecular analysis was undertaken to deter-
FIG. 3. (A and B) Representative metaphases illustrating the satellited chromosome 17 (small arrow) and the normal chromosome 17 with a grain (large arrow) hybridized to band ~13. In situ hybridization was carried out using the method described by Cannizxaro et al. (1). Slides were coated with Kodak NTB-2 and exposed for 5-6 days at 4OC. Slides were developed and fixed at 15°C and stained following the procedure of Canniszaro and Emanuel (2).
SHORT
COMMUNICATION
mine if any of the distal region of band 17~13 was absent from the satellited 17~. Genomic DNA from the patient and both parents was utilized. Southern analysis using YNZ22.1 and p144D6 in sequential hybridization to the same filter revealed the patient to be heterozygous for the former locus but apparently
FIG.
623
hemizygous for the latter (Figs. 2A and 2B). Analysis using YNZH37.3 and p144D6 on another filter indicated similar findings (Fig. 2C, data for p144D6 not shown). Simultaneous hybridization and densitometric analysis (Schwartz et al., 1988) of the filters used in Fig. 2 with a chromosome 4 probe (D4Sl) and p144D6
3-Continued
SHORT
624
13p
11.2ll.l11.221.121.3-
q 23-
13-m
oooooooooo y~e~o*oo
P :..
11.2ll.l11.2-
.
21.121.3-
0 .
COMMUNICATION
4
critical region for MDS. The genomic DNA from our patient may actually define the distal limits of the smallest region of overlap in MDS patients. As such, it should prove useful in mapping probes to the MillerDieker critical region.
p-0
l 00
ACKNOWLEDGMENTS
230 25-
normal
17
Satellited
17
FIG. 4. Schematic of the normal and satellited chromosome 17 illustrating hybridization of probe p144D6. The upper two rows of grains shown associated with the normal chromosome 17 represent hybridization to the entire p13 band.
indicated that the single band detected by probe p144D6 was representative of one copy (data not shown). To further confirm the finding of one copy of locus D17S34, in situ hybridization was carried out on metaphase spreads from the patient. Figures 3 and 4 show a representative metaphase and a schematic of the distribution of grains associated with chromosome 17, respectively. Eighteen metaphases (18.4%) were labeled on 17~13. Forty-three of 387 hybridizedgrains (11.1%) were associated with chromosome 17. Of the 43 grains associated with 17, 21 grains (48.8%) were present on ~13. Twenty of these 21 (95%) were on band p13 of the normal chromosome (Fig. 4). These 21 grains represented 5.4% of the total label. Thus, the in situ hybridization pattern was consistent with the molecular findings. Previous studies (Schwartz et aZ., 1988; vanTunien et al., 1988) found probes YNZ22.1 and YNH37.3 to be consistently absent from one chromosome 17 in their population of MDS patients. However, approximately 25% of these samepatients retained two copies of probe p144D6, the most distal marker mapped to 17~13 (Nakamura et al., 1988). This subset of patients included one MDS patient with a visible deletion of 17~13, indicating that the deletion was interstitial rather than terminal (Schwartz et al., 1988). The molecular analysis of the present patient, employing the sameprobes utilized in the MDS studies, indicated only probe p144D6 to be absent from the satellited chromosome 17. This patient can be viewed as complementary to the MDS patients who had two copies of probes p144D6 but lacked one copy of probes YNZ22.1 and YNH37.3. Clinically, our patient does not exhibit the MDS phenotype-mental retardation, lissencephaly, recognizable facial malformations-and is active, with normal growth at age 9 months. This would indicate that the absence of locus D17S34 (p144D6) does not result in MDS expression and lies distal to the
We thank Dr. Y. Nakamura, Howard Hughes Medical Institute, University of Utah, for providing probes YNZ22.1 (D17S5) and pYNH37.3 (D17S28) and Dr. M. Litt, Department of Medical Genetics, Oregon Health Sciences University, for providing probe p144D6 (D17S34). Dr. W. Krause and Mrs. C. MeadIex obtained follow-up clinical information on the family. Marc Fructman and Chi Tran provided technical assistance and Angela Parks Godfrey typed the manuscript. This work was supported in part by grants from the Self Foundation, the South Carolina Department of Mental Retardation, and a National Cancer Institute grant (CA-41763) to S.R.P.
REFERENCES 1.
2.
3.
4.
CANNIZZARO, L. A., ARONSON, M. M., AND EMANUEL, B. S. (1985). In situ hybridization and translocation breakpoint mapping, II. Two unusual t(21;22) translocations. Cytogenet. Cell Genet. 39: 173-178. CANNIZZAFLO, L. A., AND EMANUEL, B. S. (1934). An improved method for G-banding chromosomes after in situ hybridization. Cytogenet. Cell Genet. 38: 306-309. W. B., STRATTON, R. F., AND GREENBERG, F. (1984). Syndromes with lissencephaly. I. Miller-Dieker and NormanRoberts syndromes and isolated lissencephsly. Amer. J. Med. Genet. 18: 509426. DOBYNS, W. B., STRA'ITON, R. F., PARKE, J. T., GREENBERG, F., NUSSBAUM, R. F., AND LEDBE~ER, D. H. (1983). MillerDieker syndrome and monosomy 17~. J. Pedintr. 102: 662DOBYNS,
558. 5.
NAKAMURA, Y., LATHROP, M., O’CONNELL, P., LEPPERT, M., BARKER, D., WRIGHT, E., SKOLNICK, M., KONDOLEON, S., Lrrr, M., LALOUEL, J. M., AND WHITE, R. (1933). A mapped set of markers for human chromosome 17. Genomics 2: 302-309.
6.
SCHMICKEL, R. D. (1936). Contiguous gene syndromes: A component of recognizable syndromes. J. Pediutr. 109: 231-241. SCHWARTZ, C. E., MCNALLY, E., LEINWAND, L., AND SKOLNICK, M. H. (1986). Linkage of a myosin heavy chain locus to an anonymous single copy locus (D17Sl) at 17~13. Cytogenet. Cell Genet. 43: 117-120. SCHWARTZ, C. E., JOHNSON, J. P., HOLYCROSS, B., MANDEVII,LE, T. M., SEARS, T. S., GRAUL, E. A., CAREY, J. C., SCHROER, R. J., PHELAN, M. C., SZOLLAR, J., FLANNERY, D. B., AND STEVENSON, R. E. (1988). Detection of submicroscopic deletions in band 17~13 in patients with the Miller-Dieker syndrome. Amer. J. Hum. Genet. 43: 597-604. STRATTON, R. F., DOBYNS, W. B., AIRHART, S. C., AND LEDBETTER, D. H. (1984). New chromosomal syndrome: MillerDieker syndrome and monosomy 17~13. Hum. Genet. 67: 19%
7.
8.
9.
200.
10.
VANTIIINEN, P., DOBYNS, W. B., RICH, D. C., SUMMERS, K. M., ROBINSON, T. J., NAKAMUU, Y., AND LEDBEI-IER, D. H. (1933). Molecular detection of microscopic and submicroscopic deletions associated with Miller-Dieker syndrome. Amer. J. Hum. Genet. 43: 567-596.
7,621~624
(1990)
SHORT COMMUNICATION Localization of the Miller-Dieker Critical Region Is Proximal to Locus D17534 (pl44D6) in 17~13.3 CHARLES E. ScHwARTz,*,tpl SUZANNE M. SAUER,*-* ANGELA M. BROWN,* JAMES E. DIVELBISS,$ AND SHIVANAND R. PATIL* *Greenwood
Genetic Center, Greenwood, South Carolina, tDepartment of Medical Genetics, Self Memorial Greenwood, South Carolina, and *Department of Pediatrics, Division of Medical Genetics, University of Iowa Hospitals and Clinics, Iowa City, Iowa Received
November
10, 1989;
revised
March
Hospital,
21, 1990
(D17S34), was deleted in some, but not all, of these MDS patients. Schwartz et al. (1988) suggested that this finding could mean locus D17S34 either defined the distal limit of the MDS critical region or was a portion of the critical region not deleted in some of the patients. These patients, therefore, exhibited MDS because of the absence of the region containing D17S.5 and D17S28. We have studied a male patient with a satellited short arm of chromosome 17 (Fig. 1). There was no apparent absence of satellites on any of the acrocentric chromosomes (13, 14, 15, 21,22), excluding the possibility of a reciprocal translocation. This abnormality was detected prenatally by amniocentesis due to advanced
A child with normal growth and development and the abnormal karyotype 46,XY,17ps, was analyzed using molecular probes localized to 17~ 13. The results indicated the presence of two copies of the probes YNZ22.1 (Dl755) and YNH37.3 (D17528), previously shown to be deleted in all Miller-Dieker (MDS) patients studied. However, the patient was hemizygous for probe p144D6 (D17534), which is absent in approximately 75% of the MDS patients. As the patient is active at 9 months of age, with no clinical signs of MDS, the results conArm that the absence of locus D17S34 does not lead to the phenotypic expression of MDS. Furthermore, this deletion should assist in defining tbe distal limits of this contiguous gene syndrome. 8 lssoAcademic PWE,IIIC.
The Miller-Dieker syndrome (MDS) is an example of the group of disorders known as “contiguous gene syndromes” (Schmickel, 1986). MDS exhibits a combination of recognizable facial malformations, retarded growth, and lissencephaly (Dobyns et al., 1984). It is quite often associated with a deletion of chromosome band 17~13 (Dobyns et al., 1983; Stratton et al., 1984). Recently, Schwartz et al. (1988) and vanTunien et al. (1988) demonstrated that although all cases of MDS may not exhibit a visible deletion of 17p, all apparently have a submicroscopic deletion. Both of these groups found two highly polymorphic DNA probes, YNZ22.1 (D17S5) and YNH37.3 (D17S28), to be deleted in all of their MDS patients. A third probe, p144D6 ‘To whom reprint requests should be addressed at Greenwood Genetic Center, 1 Gregor Mendel Circle, Greenwood, SC 29646. * Present address: Department of Medical Genetics, Indiana University Medical Center, Indianapolis, IN.
FIG. normal 621
1. Partial chromosome
karyotypes of the patient from two cells showing 17 and satellited chromosome (arrow). osss-7543/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
622
SHORT
A.
COMMUNICATION
C.
8.
FIG. 2. Restriction fragments detected by the three chromosome 17 probes digestion; (B) p144D6 after ZizqI digestion; (C) YNH37.3 after MspI digestion. labeling of probes were done as previously described (8). Filters were sequentially p144D6 (MspI).
maternal age. Both parents were found to be chromosomally normal. On examination at 9 months of age, the patient was found to be active with good verbal and motor skills and normal growth. Except for epi-
after digestion of genomic DNA. F, father; Pt, patient; M, mother. probed with YNZ22.1 and p144D6
(A) YNZ22.1 after Southern transfers (TaqI) or YNH37.3
‘Z’oqI and and
canthal folds and broad feet, his physical examination was normal. Given the lack of any clinical signs of MDS in the patient, molecular analysis was undertaken to deter-
FIG. 3. (A and B) Representative metaphases illustrating the satellited chromosome 17 (small arrow) and the normal chromosome 17 with a grain (large arrow) hybridized to band ~13. In situ hybridization was carried out using the method described by Cannizxaro et al. (1). Slides were coated with Kodak NTB-2 and exposed for 5-6 days at 4OC. Slides were developed and fixed at 15°C and stained following the procedure of Canniszaro and Emanuel (2).
SHORT
COMMUNICATION
mine if any of the distal region of band 17~13 was absent from the satellited 17~. Genomic DNA from the patient and both parents was utilized. Southern analysis using YNZ22.1 and p144D6 in sequential hybridization to the same filter revealed the patient to be heterozygous for the former locus but apparently
FIG.
623
hemizygous for the latter (Figs. 2A and 2B). Analysis using YNZH37.3 and p144D6 on another filter indicated similar findings (Fig. 2C, data for p144D6 not shown). Simultaneous hybridization and densitometric analysis (Schwartz et al., 1988) of the filters used in Fig. 2 with a chromosome 4 probe (D4Sl) and p144D6
3-Continued
SHORT
624
13p
11.2ll.l11.221.121.3-
q 23-
13-m
oooooooooo y~e~o*oo
P :..
11.2ll.l11.2-
.
21.121.3-
0 .
COMMUNICATION
4
critical region for MDS. The genomic DNA from our patient may actually define the distal limits of the smallest region of overlap in MDS patients. As such, it should prove useful in mapping probes to the MillerDieker critical region.
p-0
l 00
ACKNOWLEDGMENTS
230 25-
normal
17
Satellited
17
FIG. 4. Schematic of the normal and satellited chromosome 17 illustrating hybridization of probe p144D6. The upper two rows of grains shown associated with the normal chromosome 17 represent hybridization to the entire p13 band.
indicated that the single band detected by probe p144D6 was representative of one copy (data not shown). To further confirm the finding of one copy of locus D17S34, in situ hybridization was carried out on metaphase spreads from the patient. Figures 3 and 4 show a representative metaphase and a schematic of the distribution of grains associated with chromosome 17, respectively. Eighteen metaphases (18.4%) were labeled on 17~13. Forty-three of 387 hybridizedgrains (11.1%) were associated with chromosome 17. Of the 43 grains associated with 17, 21 grains (48.8%) were present on ~13. Twenty of these 21 (95%) were on band p13 of the normal chromosome (Fig. 4). These 21 grains represented 5.4% of the total label. Thus, the in situ hybridization pattern was consistent with the molecular findings. Previous studies (Schwartz et aZ., 1988; vanTunien et al., 1988) found probes YNZ22.1 and YNH37.3 to be consistently absent from one chromosome 17 in their population of MDS patients. However, approximately 25% of these samepatients retained two copies of probe p144D6, the most distal marker mapped to 17~13 (Nakamura et al., 1988). This subset of patients included one MDS patient with a visible deletion of 17~13, indicating that the deletion was interstitial rather than terminal (Schwartz et al., 1988). The molecular analysis of the present patient, employing the sameprobes utilized in the MDS studies, indicated only probe p144D6 to be absent from the satellited chromosome 17. This patient can be viewed as complementary to the MDS patients who had two copies of probes p144D6 but lacked one copy of probes YNZ22.1 and YNH37.3. Clinically, our patient does not exhibit the MDS phenotype-mental retardation, lissencephaly, recognizable facial malformations-and is active, with normal growth at age 9 months. This would indicate that the absence of locus D17S34 (p144D6) does not result in MDS expression and lies distal to the
We thank Dr. Y. Nakamura, Howard Hughes Medical Institute, University of Utah, for providing probes YNZ22.1 (D17S5) and pYNH37.3 (D17S28) and Dr. M. Litt, Department of Medical Genetics, Oregon Health Sciences University, for providing probe p144D6 (D17S34). Dr. W. Krause and Mrs. C. MeadIex obtained follow-up clinical information on the family. Marc Fructman and Chi Tran provided technical assistance and Angela Parks Godfrey typed the manuscript. This work was supported in part by grants from the Self Foundation, the South Carolina Department of Mental Retardation, and a National Cancer Institute grant (CA-41763) to S.R.P.
REFERENCES 1.
2.
3.
4.
CANNIZZARO, L. A., ARONSON, M. M., AND EMANUEL, B. S. (1985). In situ hybridization and translocation breakpoint mapping, II. Two unusual t(21;22) translocations. Cytogenet. Cell Genet. 39: 173-178. CANNIZZAFLO, L. A., AND EMANUEL, B. S. (1934). An improved method for G-banding chromosomes after in situ hybridization. Cytogenet. Cell Genet. 38: 306-309. W. B., STRATTON, R. F., AND GREENBERG, F. (1984). Syndromes with lissencephaly. I. Miller-Dieker and NormanRoberts syndromes and isolated lissencephsly. Amer. J. Med. Genet. 18: 509426. DOBYNS, W. B., STRA'ITON, R. F., PARKE, J. T., GREENBERG, F., NUSSBAUM, R. F., AND LEDBE~ER, D. H. (1983). MillerDieker syndrome and monosomy 17~. J. Pedintr. 102: 662DOBYNS,
558. 5.
NAKAMURA, Y., LATHROP, M., O’CONNELL, P., LEPPERT, M., BARKER, D., WRIGHT, E., SKOLNICK, M., KONDOLEON, S., Lrrr, M., LALOUEL, J. M., AND WHITE, R. (1933). A mapped set of markers for human chromosome 17. Genomics 2: 302-309.
6.
SCHMICKEL, R. D. (1936). Contiguous gene syndromes: A component of recognizable syndromes. J. Pediutr. 109: 231-241. SCHWARTZ, C. E., MCNALLY, E., LEINWAND, L., AND SKOLNICK, M. H. (1986). Linkage of a myosin heavy chain locus to an anonymous single copy locus (D17Sl) at 17~13. Cytogenet. Cell Genet. 43: 117-120. SCHWARTZ, C. E., JOHNSON, J. P., HOLYCROSS, B., MANDEVII,LE, T. M., SEARS, T. S., GRAUL, E. A., CAREY, J. C., SCHROER, R. J., PHELAN, M. C., SZOLLAR, J., FLANNERY, D. B., AND STEVENSON, R. E. (1988). Detection of submicroscopic deletions in band 17~13 in patients with the Miller-Dieker syndrome. Amer. J. Hum. Genet. 43: 597-604. STRATTON, R. F., DOBYNS, W. B., AIRHART, S. C., AND LEDBETTER, D. H. (1984). New chromosomal syndrome: MillerDieker syndrome and monosomy 17~13. Hum. Genet. 67: 19%
7.
8.
9.
200.
10.
VANTIIINEN, P., DOBYNS, W. B., RICH, D. C., SUMMERS, K. M., ROBINSON, T. J., NAKAMUU, Y., AND LEDBEI-IER, D. H. (1933). Molecular detection of microscopic and submicroscopic deletions associated with Miller-Dieker syndrome. Amer. J. Hum. Genet. 43: 567-596.