Y-derived sequence detected in minute chromosome; by polymerase chain reaction and iB situ hybridization Tsukahara M, Matsuura S, Kishi F, Fisher LK, Stock AD, Izumikawa Y, Naritomi K, Kajii T. Y-derived sequence detected in minute chromosomes by polymerase chain reaction and in siru hybridization. Clin Genet 1992: 42: 80-83. A 10-year-old girl and a 10-month-old girl, both with ambiguous genitalia, were found to have 45,X/46,X,mar and 45,X/46,X,r(?) mosaicism. The marker chromosomes in both girls were very small. Polymerase chain reaction, with synthetic oligonucleotide primers from Y-specific DNA sequences pY-80 and pY53.3 containing the sex-determining region Y(SRY), proved the marker chromosomes to contain the Y short arm material. In situ hybridization with probe pY-80 confirmed that the marker chromosomes included the Y short arms. These findings, together with ambiguous genitalia in the girls, indicate that the marker chromosomes include the testis-determining factor gene.
Masato Tsukahara', Sbinya Matsuura', Furnio Kishi', Lynda K. Fithe?, A. Dean Stack), Yoshinori Izumikawa4, Kenji Naritomi' and Tadashi Kajii' Departments of Pediatrics, 'Yamaguchi Universrty School of Medicine, Yamaguchi. arid 'Ryukyu University schod of Medicine, Okinawa. Japan and %ity of Hope National Medical Center, Caliiomia, and 'Department of Pathology and Laboratory Medicine. University of Nevada School of Medicine Genetics Network. Cytogenetics Laboratory, Nevada USA
Key words: in situ hybridization - marker chrom o m e polymerase chain reaction Y-specific DNA sequences
-
-
Dr Masato Tsukahara, Department of Pediatrics, Yamaguchi University School of Medicine, Ube. Yarnaguchi, 755 Japan Received 3 January, accepted for publication 3 April 1992
A minute chromosome of unknown origin poses a counseling dilemma in a prepubertal girl with ambiguous genitalia. The combined use of various chromosome banding methods and chromosomespecific DNA probes now allows precise identification of such minute chromosomes (Miiller et al. 1987, Lin et al. 1990, Cooper et al. 1991, Callen et al. 1991). We describe here two prepubertal girls with a minute chromosome, studied using polymerase chain reaction and in situ hybridization analysis with Y-specific probes. Material amd methods Patients
Patient I (89112), a Mexican girl, was born to a healthy mother. Ambiguity of the genitalia was noted, and the midwife was unable to assign a sex. At 3 months of age, erections were noted. The mother noted that the phallus felt like a penis. A gonadal biopsy at age 11 months revealed immature testes. The baby was assigned a female name 80
but was raised ambiguously because the family felt the child was a male. When first seen by one of us (L.K.F.) at age 14 months, she was small and had a prominent forehead, a heart-shaped face and a broad neck. There was a shawl appearance to the labio-scrota1 folds, which were fused posteriorly. The phallus measured 2.5 an long and 0.9 cm wide (at the glans) and had a severe chordee. The labio-scrota1 folds were rugated. There was a single orifice on the perineum at the junction of the phallus and perineum. Small (1 cm)oblong gonads were palpable high in the labio-scrota1folds. A genitogram indicated that the vagina was absent. The utriculus arose from the prostatic portion of the urethra. The anterior urethra was approximately 5 cm in length. Chromosome analysis on PHA-stimulated peripheral blood lymphocytes revealed a 45,X/ 46,X,min (43%/57%) karyotype. The minute (min) marker chromosome was so tiny that it was uncertain whether it was a ring. The serum LH was 3.2 mIU/ml, and FSH was
Minute Y use of short arm probes
less than 1.6 mIU/ml. In a 24-h urine, etiocholanolone was less than 0.1 mg, and androsterone 0.1 mg. The patient underwent a repeat endocrine evaluation prior to bilateral gonadectomy at age 19 months. LH was 1.9 mIU/ml; FSH,2.0 mIU/ ml; androstenedione, 11 ng/dl; testosterone 5.4 ng/ dl; DHEAS, 30 ng/dl. After HCG (10000 units over 3 days) the androstenedione was 26 ng/dl and the testosterone 275 ng/dl. A complete cardiac evaluation was normal. She had only one kidney. Bilateral gonadectomy and clitoral resection were camed out, and karyotypes were done on the gonads and genital skin. The right and left gonads had a greater preponderance of the 45,X cell line (right: 17 of 18 cells hypodiploid and one with the marker; left: 11 of 12 cells hypodiploid and one with the marker). The genital skin fibroblasts were 45,X in 19 of 21 cells; the remaining two cells contained 46,X, plus the marker. H-Y antigen testing was negative in all tissues. Chromosomes froin peripheral blood reevaluated at age 6 years were 45,X/46,X,mar (14%/86%) (Fig. la). ..
'*
a
_.I
'
.
Fig. 1. G-banded metaphase cells from patient 1 (a) and patient 2 (b). Arrow indicates the marker chromosome. Insets indicate the magnified marker chromosome.
1 1 2 3 4 5 6 7 8
.
0.7-
**)*I,
Fig. 2. Left: Electrophoretic analysis of PCR products using pY-80. Lane 1: patient I ; lane 2: patient 2; lanes 3-5: males; lanes 6-8: females. Right: Southern blot analysis of the gel with a '*P-labeled Pstl-EcoRI fragment probe. The size of the PCR product is marked in kb.
The child is now 10 years old and seems to be a well-adjusted girl with a normaf IQ. She is extremely short and has followed the growth pattern of girls with the Turner syndrome. Patient 2 (89124), a Japanese girl, was born after a 34-week pregnancy, with premature rupture of the placental membranes, to a 27-year-old primigravida mother and a 29-year-old father, both healthy and nonconsanguineous. At birth, she weighed 1574 g (- 1.5 S.D.) and measured 39 cm (-2.2 S.D.). She was placed in an incubator for 4 weeks. She was referred to us at age 3 months because of an enlarged clitoris. She then weighed 5450 g (-2.4 S.D.), measured 57 an (-4.2 S.D.), and her She head circumference was 40.2 cm (+0.8 S.D.). was dolichocephalic. She had a short nose, prominent ears, a right cupped ear, a high-arched palate, a short neck with redundant skin, multiple hemangiomas on the body, an umbilical hernia and hypertrophy of the clitoris. Her serum FSH level was elevated at 22.3 mIU/ ml (normal range, 8.8 f5.2 mIU/ml). Normal laboratory tests included serum LH, plasma testosterone (HCG stimulation), serum estrogen, serum progesterone, serum 11-OHCS, serum 17-OHP, T3, T4, TSH,ACT'H, and urinary 17-ketosteroids. Intravenous pyelography revealed left ureterocele. Chromosome analysis on PHA-stimulated peripheral blood lymphocytes revealed 45,X/46,X,r(?) mosaicism (84%/ 16%) (Fig. 1b). She is now 10 months, with a D.Q. of 113. 81
Tsukahara et al. DNA extraction
Total genomic DNA was extracted from EB virustransformed lymphoblastoid cell lines of patients 1 and 2 by the method of Blin & Stafford (1976), with a slight modification. DNA probes
PY-80 and pY53.3, both Y chromosome-specific DNA sequences, were used in this study. PY-80, derived from Ypl1.2-Ypter, was isolated from a flow-sorted,human Y chromosome library (ATCC No. 57715), and is 4.6 kb long (Tsukahara et al. 1990). Probe pY53.3 contained the sexdetermining region Y (SRY), a candidate for the testis-determining gene (TDF). It is 2.1 kb long, and is located within the pseudoautosomal boundary (Sinclair et al. 1990).
a
-
- . -
.. . . -.
_.__ .
Polymerase chain reaction (PCR) and Southern blot analysis
Oligomers used for PCR were synthesized on an Applied Biosystem 381A DNA Synthesizer. The sequences of oligomers from pY-80 were Y 15’ATGACCCTAGAACCACTGGA-3’ (upstream), and Y2:5’-CGGlTGCTGAAAGATACAAG-3’ (downstream). The sequences of oligomers from pY53.3 were 5’-GAATATTCCCGCTCTCCGGA-
b
c
Fig. 4. In situ hybridization with pY-80 probe from patient 1
(a) and patient 2 (b). Arrows indicate the marker chromosomes.
3 0.47-
Fig. 3. Electrophoretic analysis of PCR products using pY53.3. Lane 1: patient 1; lane 2: patient 2; lanes 3-5: males; lanes 6
and 7: females.
82
3‘ (upstream), and 5’-AGCTGGTGCTCCATTCTTGA-3’ (downstream). The reaction mixture consisted of 50 mM KCI, 10 mM Tris-HC1 (pH 8.3), 1.5 mM MgCl,, 0.01% gelatin, 5 pmol of each primer, 0.2 mM of each of the four deoxynucleotide triphosphates (dATP, dCTP, dTTP, and dGTP), 5 units of Taq polymerase and sample for a total volume of 50 p1. The amplification reaction was performed in a program temperature control system (PC600) (Astec Co. Fukuoka, Japan). The samples were subjected to 30 cycles of denaturation at 94°C for 1.5 min, annealing at 50°C for 2 min and extension at 70°C for 3 min. The amplified product was electrophoresed on 1% agarose gel. Southern blotting for amplified product of pY-80 was further camed out using 32P-labeled PstI-EcoRI fragment of pY-80 as a probe.
In situ chromosome hybridization EB virus-transformed cell lines were treated with 5-bromodeoxyuridine (10 pg/ml) for 5 h prior to
Minute Y: use of short arm probes harvest, and colcemid (0.03 pg/ml) was added for 2 h prior to harvest. Chromosome slides were prepared by the conventional air-dried method. In situ hybridization using probe pY-80 was performed by the methods described elsewhere (Tsukahara & Yoshida 1989). Results PCR and Southern blot analysis
Electrophoretic analysis of the PCR products using pY-80 from patients 1 and 2 revealed a 0.7 kb band with male specificity (Fig. 2, left). Southern blot analysis of the gel with a 32P-labeledPsrI-EcoRI fragment of pY-80 also revealed male specificity (Fig. 2, right). Electrophoretic analysis of the PCR products using pY53.3 from patients 1 and 2 revealed a 0.47 kb band with male specificity (Fig. 3).
chromosome in the baby was derived from the Y chromosome. Southern blot analysis or in situ hybridization with Y-specific DNA probes has been used for establishing the Y-chromosome origin of a minute chromosome in patients with Turner syndrome with 45,X/46,X,mar (Muller et al. 1987, Lin et al. 1990) or 45,X/46,X,dic?(Y) mosaicism (Cooper et al. 1991). Turner syndrome patients with non-fluorescent Y chromosome have a high risk of developing gonadoblastoma (Scully 1970).Therefore, it is clinically important to confirm the origin (X or Y chromosome) of a minute marker chromosome. The PCR with probes pY-80 and pY53.3 as described by us is simple and rapid and would be useful for the identification of Y chromosomal abnormalities involving the short arm.
Acknowledgement In sit0 hybridization with pY-80
Patient I: A total of 50 metaphase cells with wellspread and high-quality banded chromosomes were analyzed. Overall, 145 grains were observed, of which 24 (16.6%) were located on the marker chromosome (Fig. 421). Patient 2: A total of 51 metaphase cells with marker chromosome were analyzed. Overall, 177 grains were observed, of which 15 (8.5Y0) were located on the marker chromosome (Fig. 4b). Discussion
The two girls we describe here both had ambiguous genitalia, with an enlarged clitoris and a 45,X/ 46,X,min karyotype. The minute chromosomes were difficult to analyze with conventional cytogenetic methods. Electrophoretic analysis of the PCR products from the two girls indicated that both had a Y short arm segment, corresponding to pY-80 and pY53.3. Southern blot analysis with a PstI-EcoRI fragment of pY-80 as a probe further supported the finding. In situ hybridization chromosome analysis with pY-80 as a probe indicated that the minute chromosomes contained the pY-80 segment. These findings together implied that the minute chromosomes in the two girls were derived from the Y chromosome. Muller et al. (1987) reported a newborn baby with ambiguous genitalia who had a 45,X/ 46,X,mar karyotype. Southern blot analysis using a Y-specific probe, Y-190, proved that the marker
This work was supported in part by a Grant-in-Aid (No. 01570536) from the Ministry of Education, Science and Culture of Japan.
References Blin N, Stafford DW. A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res 1976: 3: 2303-2308. Callen DF, Eyre HJ, Ringenbergs ML, Freemantle CJ, Woodroffe P,Haan EA. Chromosomal origin of small ring marker chromosomes in man: characterization by molecular genetics. Am J Hum Genet 1991: 48: 769-782. Cooper C, Crolla JA, Laister C, Johnston DI, Cooke P. An investigation of ring and dicentric chromosomes found in three Turner’s syndrome patients using DNA analysis and in siru hybridization with X and Y chromosome specific probes. J Med Genet 1991: 28: 6-9. Lin CC, Meyne J, Sasi R, Bowen P, Unger T, Tainaka T, Hadro TA, Hoo JJ. Determining the origins and the structural aberrations of small marker chromosomes in two cases of 45,XI 46,X,+mar by use of chromosome-specific DNA probes. Am J Med Genet 1990: 37: 71-78. Miiller U, Donlon TA, Kunkel SM, Lalande M, Latt SA. Y190, a DNA probe for the sensitive detection of Y-derived marker chromosomes and mosaicism. Hum Genet 1987: 75: 109-1 13. Scully RE. Gonadoblastoma. A review of 74 cases. Cancer 1970 25: 1340-1355. Sinclair AH, Berta P,Palmer MS, Hawkins JR, Griffiths BL, Smith MJ, Foster JW, Frischauf A-M, Lovell-Badge R, Goodfellow PN. A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif. Nature 1990: 346 240-244. Tsukahara M. Yoshida A. Chromosomal assignment of the alcohol dehydrogenase cluster locus to human chromosome 4q21-23 by in siru hybridization. Genomics 1989: 46: 218-220. Tsukahara M, Matsuura S, Kishi F, Yoshida A. Kajii T. Isolation of a human Y chromosomal DNA sequence and its clinical application. Jpn J Hum Genet 1990: 35: 331-339.
83
Masato Tsukahara', Sbinya Matsuura', Furnio Kishi', Lynda K. Fithe?, A. Dean Stack), Yoshinori Izumikawa4, Kenji Naritomi' and Tadashi Kajii' Departments of Pediatrics, 'Yamaguchi Universrty School of Medicine, Yamaguchi. arid 'Ryukyu University schod of Medicine, Okinawa. Japan and %ity of Hope National Medical Center, Caliiomia, and 'Department of Pathology and Laboratory Medicine. University of Nevada School of Medicine Genetics Network. Cytogenetics Laboratory, Nevada USA
Key words: in situ hybridization - marker chrom o m e polymerase chain reaction Y-specific DNA sequences
-
-
Dr Masato Tsukahara, Department of Pediatrics, Yamaguchi University School of Medicine, Ube. Yarnaguchi, 755 Japan Received 3 January, accepted for publication 3 April 1992
A minute chromosome of unknown origin poses a counseling dilemma in a prepubertal girl with ambiguous genitalia. The combined use of various chromosome banding methods and chromosomespecific DNA probes now allows precise identification of such minute chromosomes (Miiller et al. 1987, Lin et al. 1990, Cooper et al. 1991, Callen et al. 1991). We describe here two prepubertal girls with a minute chromosome, studied using polymerase chain reaction and in situ hybridization analysis with Y-specific probes. Material amd methods Patients
Patient I (89112), a Mexican girl, was born to a healthy mother. Ambiguity of the genitalia was noted, and the midwife was unable to assign a sex. At 3 months of age, erections were noted. The mother noted that the phallus felt like a penis. A gonadal biopsy at age 11 months revealed immature testes. The baby was assigned a female name 80
but was raised ambiguously because the family felt the child was a male. When first seen by one of us (L.K.F.) at age 14 months, she was small and had a prominent forehead, a heart-shaped face and a broad neck. There was a shawl appearance to the labio-scrota1 folds, which were fused posteriorly. The phallus measured 2.5 an long and 0.9 cm wide (at the glans) and had a severe chordee. The labio-scrota1 folds were rugated. There was a single orifice on the perineum at the junction of the phallus and perineum. Small (1 cm)oblong gonads were palpable high in the labio-scrota1folds. A genitogram indicated that the vagina was absent. The utriculus arose from the prostatic portion of the urethra. The anterior urethra was approximately 5 cm in length. Chromosome analysis on PHA-stimulated peripheral blood lymphocytes revealed a 45,X/ 46,X,min (43%/57%) karyotype. The minute (min) marker chromosome was so tiny that it was uncertain whether it was a ring. The serum LH was 3.2 mIU/ml, and FSH was
Minute Y use of short arm probes
less than 1.6 mIU/ml. In a 24-h urine, etiocholanolone was less than 0.1 mg, and androsterone 0.1 mg. The patient underwent a repeat endocrine evaluation prior to bilateral gonadectomy at age 19 months. LH was 1.9 mIU/ml; FSH,2.0 mIU/ ml; androstenedione, 11 ng/dl; testosterone 5.4 ng/ dl; DHEAS, 30 ng/dl. After HCG (10000 units over 3 days) the androstenedione was 26 ng/dl and the testosterone 275 ng/dl. A complete cardiac evaluation was normal. She had only one kidney. Bilateral gonadectomy and clitoral resection were camed out, and karyotypes were done on the gonads and genital skin. The right and left gonads had a greater preponderance of the 45,X cell line (right: 17 of 18 cells hypodiploid and one with the marker; left: 11 of 12 cells hypodiploid and one with the marker). The genital skin fibroblasts were 45,X in 19 of 21 cells; the remaining two cells contained 46,X, plus the marker. H-Y antigen testing was negative in all tissues. Chromosomes froin peripheral blood reevaluated at age 6 years were 45,X/46,X,mar (14%/86%) (Fig. la). ..
'*
a
_.I
'
.
Fig. 1. G-banded metaphase cells from patient 1 (a) and patient 2 (b). Arrow indicates the marker chromosome. Insets indicate the magnified marker chromosome.
1 1 2 3 4 5 6 7 8
.
0.7-
**)*I,
Fig. 2. Left: Electrophoretic analysis of PCR products using pY-80. Lane 1: patient I ; lane 2: patient 2; lanes 3-5: males; lanes 6-8: females. Right: Southern blot analysis of the gel with a '*P-labeled Pstl-EcoRI fragment probe. The size of the PCR product is marked in kb.
The child is now 10 years old and seems to be a well-adjusted girl with a normaf IQ. She is extremely short and has followed the growth pattern of girls with the Turner syndrome. Patient 2 (89124), a Japanese girl, was born after a 34-week pregnancy, with premature rupture of the placental membranes, to a 27-year-old primigravida mother and a 29-year-old father, both healthy and nonconsanguineous. At birth, she weighed 1574 g (- 1.5 S.D.) and measured 39 cm (-2.2 S.D.). She was placed in an incubator for 4 weeks. She was referred to us at age 3 months because of an enlarged clitoris. She then weighed 5450 g (-2.4 S.D.), measured 57 an (-4.2 S.D.), and her She head circumference was 40.2 cm (+0.8 S.D.). was dolichocephalic. She had a short nose, prominent ears, a right cupped ear, a high-arched palate, a short neck with redundant skin, multiple hemangiomas on the body, an umbilical hernia and hypertrophy of the clitoris. Her serum FSH level was elevated at 22.3 mIU/ ml (normal range, 8.8 f5.2 mIU/ml). Normal laboratory tests included serum LH, plasma testosterone (HCG stimulation), serum estrogen, serum progesterone, serum 11-OHCS, serum 17-OHP, T3, T4, TSH,ACT'H, and urinary 17-ketosteroids. Intravenous pyelography revealed left ureterocele. Chromosome analysis on PHA-stimulated peripheral blood lymphocytes revealed 45,X/46,X,r(?) mosaicism (84%/ 16%) (Fig. 1b). She is now 10 months, with a D.Q. of 113. 81
Tsukahara et al. DNA extraction
Total genomic DNA was extracted from EB virustransformed lymphoblastoid cell lines of patients 1 and 2 by the method of Blin & Stafford (1976), with a slight modification. DNA probes
PY-80 and pY53.3, both Y chromosome-specific DNA sequences, were used in this study. PY-80, derived from Ypl1.2-Ypter, was isolated from a flow-sorted,human Y chromosome library (ATCC No. 57715), and is 4.6 kb long (Tsukahara et al. 1990). Probe pY53.3 contained the sexdetermining region Y (SRY), a candidate for the testis-determining gene (TDF). It is 2.1 kb long, and is located within the pseudoautosomal boundary (Sinclair et al. 1990).
a
-
- . -
.. . . -.
_.__ .
Polymerase chain reaction (PCR) and Southern blot analysis
Oligomers used for PCR were synthesized on an Applied Biosystem 381A DNA Synthesizer. The sequences of oligomers from pY-80 were Y 15’ATGACCCTAGAACCACTGGA-3’ (upstream), and Y2:5’-CGGlTGCTGAAAGATACAAG-3’ (downstream). The sequences of oligomers from pY53.3 were 5’-GAATATTCCCGCTCTCCGGA-
b
c
Fig. 4. In situ hybridization with pY-80 probe from patient 1
(a) and patient 2 (b). Arrows indicate the marker chromosomes.
3 0.47-
Fig. 3. Electrophoretic analysis of PCR products using pY53.3. Lane 1: patient 1; lane 2: patient 2; lanes 3-5: males; lanes 6
and 7: females.
82
3‘ (upstream), and 5’-AGCTGGTGCTCCATTCTTGA-3’ (downstream). The reaction mixture consisted of 50 mM KCI, 10 mM Tris-HC1 (pH 8.3), 1.5 mM MgCl,, 0.01% gelatin, 5 pmol of each primer, 0.2 mM of each of the four deoxynucleotide triphosphates (dATP, dCTP, dTTP, and dGTP), 5 units of Taq polymerase and sample for a total volume of 50 p1. The amplification reaction was performed in a program temperature control system (PC600) (Astec Co. Fukuoka, Japan). The samples were subjected to 30 cycles of denaturation at 94°C for 1.5 min, annealing at 50°C for 2 min and extension at 70°C for 3 min. The amplified product was electrophoresed on 1% agarose gel. Southern blotting for amplified product of pY-80 was further camed out using 32P-labeled PstI-EcoRI fragment of pY-80 as a probe.
In situ chromosome hybridization EB virus-transformed cell lines were treated with 5-bromodeoxyuridine (10 pg/ml) for 5 h prior to
Minute Y: use of short arm probes harvest, and colcemid (0.03 pg/ml) was added for 2 h prior to harvest. Chromosome slides were prepared by the conventional air-dried method. In situ hybridization using probe pY-80 was performed by the methods described elsewhere (Tsukahara & Yoshida 1989). Results PCR and Southern blot analysis
Electrophoretic analysis of the PCR products using pY-80 from patients 1 and 2 revealed a 0.7 kb band with male specificity (Fig. 2, left). Southern blot analysis of the gel with a 32P-labeledPsrI-EcoRI fragment of pY-80 also revealed male specificity (Fig. 2, right). Electrophoretic analysis of the PCR products using pY53.3 from patients 1 and 2 revealed a 0.47 kb band with male specificity (Fig. 3).
chromosome in the baby was derived from the Y chromosome. Southern blot analysis or in situ hybridization with Y-specific DNA probes has been used for establishing the Y-chromosome origin of a minute chromosome in patients with Turner syndrome with 45,X/46,X,mar (Muller et al. 1987, Lin et al. 1990) or 45,X/46,X,dic?(Y) mosaicism (Cooper et al. 1991). Turner syndrome patients with non-fluorescent Y chromosome have a high risk of developing gonadoblastoma (Scully 1970).Therefore, it is clinically important to confirm the origin (X or Y chromosome) of a minute marker chromosome. The PCR with probes pY-80 and pY53.3 as described by us is simple and rapid and would be useful for the identification of Y chromosomal abnormalities involving the short arm.
Acknowledgement In sit0 hybridization with pY-80
Patient I: A total of 50 metaphase cells with wellspread and high-quality banded chromosomes were analyzed. Overall, 145 grains were observed, of which 24 (16.6%) were located on the marker chromosome (Fig. 421). Patient 2: A total of 51 metaphase cells with marker chromosome were analyzed. Overall, 177 grains were observed, of which 15 (8.5Y0) were located on the marker chromosome (Fig. 4b). Discussion
The two girls we describe here both had ambiguous genitalia, with an enlarged clitoris and a 45,X/ 46,X,min karyotype. The minute chromosomes were difficult to analyze with conventional cytogenetic methods. Electrophoretic analysis of the PCR products from the two girls indicated that both had a Y short arm segment, corresponding to pY-80 and pY53.3. Southern blot analysis with a PstI-EcoRI fragment of pY-80 as a probe further supported the finding. In situ hybridization chromosome analysis with pY-80 as a probe indicated that the minute chromosomes contained the pY-80 segment. These findings together implied that the minute chromosomes in the two girls were derived from the Y chromosome. Muller et al. (1987) reported a newborn baby with ambiguous genitalia who had a 45,X/ 46,X,mar karyotype. Southern blot analysis using a Y-specific probe, Y-190, proved that the marker
This work was supported in part by a Grant-in-Aid (No. 01570536) from the Ministry of Education, Science and Culture of Japan.
References Blin N, Stafford DW. A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res 1976: 3: 2303-2308. Callen DF, Eyre HJ, Ringenbergs ML, Freemantle CJ, Woodroffe P,Haan EA. Chromosomal origin of small ring marker chromosomes in man: characterization by molecular genetics. Am J Hum Genet 1991: 48: 769-782. Cooper C, Crolla JA, Laister C, Johnston DI, Cooke P. An investigation of ring and dicentric chromosomes found in three Turner’s syndrome patients using DNA analysis and in siru hybridization with X and Y chromosome specific probes. J Med Genet 1991: 28: 6-9. Lin CC, Meyne J, Sasi R, Bowen P, Unger T, Tainaka T, Hadro TA, Hoo JJ. Determining the origins and the structural aberrations of small marker chromosomes in two cases of 45,XI 46,X,+mar by use of chromosome-specific DNA probes. Am J Med Genet 1990: 37: 71-78. Miiller U, Donlon TA, Kunkel SM, Lalande M, Latt SA. Y190, a DNA probe for the sensitive detection of Y-derived marker chromosomes and mosaicism. Hum Genet 1987: 75: 109-1 13. Scully RE. Gonadoblastoma. A review of 74 cases. Cancer 1970 25: 1340-1355. Sinclair AH, Berta P,Palmer MS, Hawkins JR, Griffiths BL, Smith MJ, Foster JW, Frischauf A-M, Lovell-Badge R, Goodfellow PN. A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif. Nature 1990: 346 240-244. Tsukahara M. Yoshida A. Chromosomal assignment of the alcohol dehydrogenase cluster locus to human chromosome 4q21-23 by in siru hybridization. Genomics 1989: 46: 218-220. Tsukahara M, Matsuura S, Kishi F, Yoshida A. Kajii T. Isolation of a human Y chromosomal DNA sequence and its clinical application. Jpn J Hum Genet 1990: 35: 331-339.
83
