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MOLECULAR ANALYSIS OF HYPOSPADIAS IN A BOY WITH DICENTRIC Y CHROMOSOME
0022-5347/01/1654-1244/0 THE JOURNAL OF UROLOGY® Copyright © 2001 by AMERICAN UROLOGICAL ASSOCIATION, INC.®
Vol. 165, 1244 –1245, April 2001 Printed in U.S.A.
MOLECULAR ANALYSIS OF HYPOSPADIAS IN A BOY WITH DICENTRIC Y CHROMOSOME YOSHIYUKI KOJIMA, YUTARO HAYASHI, YOSHIMASA YANAI, KEIICHI TOZAWA, SHOICHI SASAKI AND KENJIRO KOHRI From the Department of Urology, Nagoya City University Medical School, Nagoya, Japan KEY WORDS: urethra; hypospadias; abnormalities; Y chromosome; in situ hybridization; fluorescence
Hypospadias is the most common abnormality of the external genitalia in males. Chromosome abnormalities are common in patients with hypospadias. At our department during the 20-year period from 1979 to 1999, 400 patients underwent hypospadias repair, of whom 22 (6%) were identified with chromosomal anomalies. The dicentric Y chromosome has been reported in males with hypospadias but to our knowledge it has hardly been studied at the molecular level. In our patient with hypospadias a dicentric Y chromosome was analyzed by fluorescence in situ hybridization (FISH) with a probe for the sex determining region Y (SRY) and deleted in azoospermia (DAZ). Clinical and molecular findings are discussed.
no DAZ sequence in any chromosome. At age 2 years 1-stage urethroplasty was performed to correct hypospadias. The patient now urinates normally. DISCUSSION
Many studies have shown that the Y chromosome has a fundamental role in testis differentiation and spermatogenesis. The SRY gene is located in the vicinity of the short arm pseudo-autosomal region. Sex determination primarily depends on the presence or absence of the SRY gene. PCR of the DNA in our patient confirmed the presence of the SRY se-
CASE REPORT
A 1-month-old phenotypic male infant was referred to our hospital because of abnormality of the external genitalia. He was born weighing 2,296 gm. after 41 weeks of gestation with a normal birth. Physical examination demonstrated penile hypospadias with the urethral meatus in the posterior third of the shaft. Each testicle had descended into the scrotum. Chromosome analysis using G and Q banding showed a karyotype of 46,X⫹marker. Polymerase chain reaction (PCR) was performed to detect sequences from the SRY and DAZ sequences in peripheral blood as well as provide evidence of SRY but absent DAZ in this case (fig. 1). To confirm that the abnormal chromosome was a Y as well as the presence of SRY but absence of DAZ FISH was done with 4 biotinylated Y chromosome specific DNA probes, including DYZ1 (Yq specific repeat sequence), DYZ3 (Y centromere specific repeat sequence), SRY and DAZ. The probe for DYZ3 and SRY hybridized to the 2 regions of the abnormal chromosome but DYZ1 and DAZ did not hybridize to any region (fig. 2). Based on this evidence we proved that the marker chromosome was a dicentric Y chromosome, and this patient has 2 copies of the SRY sequence in the duplicated short arm and Accepted for publication November 22, 2000.
FIG. 1. Analysis of SRY and DAZ sequences in peripheral blood lymphocytes by PCR amplification. 1, male control. 2, female control. 3, in patient amplified 240 bp SRY fragment was present but 124 bp DAZ fragment was absent.
FIG. 2. Staining by FISH analysis. A, using SRY probe Yp11.3 (red signal), DXZ1 probe X centromere specific repeat sequence (yellow signal) and DYZ1 Yq12 (green signal) demonstrates 2 red, 1 yellow and no green signals. dic, dicentric Y. B, using DAZ probe Yq11.23 and DYZ3 probe (Y centromere specific repeat sequence showed no red and 2 green signals. 1244
MOLECULAR ANALYSIS OF HYPOSPADIAS WITH DICENTRIC Y CHROMOSOME
quence. Individuals who have a dicentric Y with long arm break points are expected to have 2 copies of the SRY sequence in the duplicated short arm.1, 2 FISH was most useful and informative for detecting the SRY sequence and localizing the signal with ease. In fact, it proved that our patient had duplication of the SRY sequence. Tuck-Muller et al reviewed the records of patients with the dicentric Y chromosome and hypospadias, which was noted in 9 of 20 phenotypic males (45%) with long arm break points but not in the 11 in the short arm break point group.2 Our patient had only a long arm break point. Although there is no proof of duplication of the SRY sequence in the patients of Tuck-Muller et al duplication of the SRY sequence may also affect urethral development. Recently 2 candidate genes for azoospermia have been cloned from the azoospermic factor locus on the long arm of the Y chromosome, of which 1 is DAZ. Some infertile men with azoospermia or severe oligospermia have small deletions in regions of the Y chromosome.3 PCR and FISH of the DNA in our patient confirmed absent DAZ. Since there is a high possibility that in our patient future fertility may be impaired because of the absent DAZ sequence, regular longterm followup is necessary.
1245
centromeres of 2 chromosomal segments (different chromosomes or sister chromatids from the same chromosome) fuse, resulting in the loss of genetic material not attached to the centromere. The most common dicentric chromosomes are X and Y. In hypospadias dicentric Y chromosomes have not been widely reported and their unique molecular findings have been studied even less. In the current case of hypospadias the authors used FISH to identify a dicentric Y chromosome with 2 copies of the SRY sequence on the duplicated short arm. In addition, the DAZ gene on the missing long arm segment and associated with male factor infertility is lacking in this child and may have profound implications on his future fertility. Understanding the molecular basis of hypospadias is in its infancy. Case reports such as this should stimulate us to study this ever increasingly common anomaly. In this child a chromosomal rearrangement results in duplication of some genetic material that is involved in gender and perhaps urethral development, and the loss of genetic material that may portend future infertility. The parsimony of this scenario is intriguing but the details of the mechanism leading to this scenario are subject to hypothesis and conjecture, and are not understood to any significant degree. The food for thought and future study is there for the asking and we should seize it so that we expand our knowledge base to beyond the technical. Lane S. Palmer Schneider Children’s Hospital Long Island Jewish Medical Center New Hyde Park, New York
REFERENCES
1. Genuardi, M., Bardoni, B., Floridia, G. et al: Dicentric chromosome Y associated with Leydig cell agenesis and sex reversal. Clin Genet, 47: 38, 1995 2. Tuck-Muller, C. M., Chen, H., Martinez, J. E. et al: Isodicentric Y chromosome: cytogenetic, molecular and clinical studies and review of the literature. Hum Genet, 96: 119, 1995 3. Reijo, R., Lee, T.-Y., Salo, P. et al: Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA-binding protein gene. Nat Genet, 10: 383, 1995 EDITORIAL COMMENT As pediatric urologists, our hypospadias knowledge base continues to grow regarding repair techniques, surgical timing to account for psychological child development and anesthetic considerations. However, the embryological and genetic basis for this anomaly remains rudimentary. The authors present molecular findings in a single case of hypospadias that stimulates those of us interested not only in hypospadias, but also in the molecular basis of congenital anomalies. Dicentric chromosomes are uncommon occurrences in which the
REPLY BY AUTHORS The progress in molecular biology in recent years has helped us to understand several diseases at the gene level, and put to practical use the findings on the mechanism and genetic diagnosis. In pediatric urology as well, several researchers and clinicians have been examining the molecular basis of various disorders, which will enable clinicians to offer more accurate diagnosis and counseling to afflicted patients and their families. Although several investigators have studied the cause of hypospadias, the exact cause remains unknown. Known causes include decreased androgen sensitivity, 5␣-reductase deficiency and chromosomal abnormalities. Since the cause of hypospadias is probably polygenic, it will be difficult to elucidate the mechanism of this disorder completely. We described a boy with hypospadias who had a dicentric Y chromosome. Molecular analysis using PCR and FISH provided evidence of the presence of short arm sequences, including SRY, their duplication and deletion of distal long arm sequences, including DAZ, in our patient. We hope that the molecular findings in this study help to expand our knowledge concerning the cause of hypospadias.
Vol. 165, 1244 –1245, April 2001 Printed in U.S.A.
MOLECULAR ANALYSIS OF HYPOSPADIAS IN A BOY WITH DICENTRIC Y CHROMOSOME YOSHIYUKI KOJIMA, YUTARO HAYASHI, YOSHIMASA YANAI, KEIICHI TOZAWA, SHOICHI SASAKI AND KENJIRO KOHRI From the Department of Urology, Nagoya City University Medical School, Nagoya, Japan KEY WORDS: urethra; hypospadias; abnormalities; Y chromosome; in situ hybridization; fluorescence
Hypospadias is the most common abnormality of the external genitalia in males. Chromosome abnormalities are common in patients with hypospadias. At our department during the 20-year period from 1979 to 1999, 400 patients underwent hypospadias repair, of whom 22 (6%) were identified with chromosomal anomalies. The dicentric Y chromosome has been reported in males with hypospadias but to our knowledge it has hardly been studied at the molecular level. In our patient with hypospadias a dicentric Y chromosome was analyzed by fluorescence in situ hybridization (FISH) with a probe for the sex determining region Y (SRY) and deleted in azoospermia (DAZ). Clinical and molecular findings are discussed.
no DAZ sequence in any chromosome. At age 2 years 1-stage urethroplasty was performed to correct hypospadias. The patient now urinates normally. DISCUSSION
Many studies have shown that the Y chromosome has a fundamental role in testis differentiation and spermatogenesis. The SRY gene is located in the vicinity of the short arm pseudo-autosomal region. Sex determination primarily depends on the presence or absence of the SRY gene. PCR of the DNA in our patient confirmed the presence of the SRY se-
CASE REPORT
A 1-month-old phenotypic male infant was referred to our hospital because of abnormality of the external genitalia. He was born weighing 2,296 gm. after 41 weeks of gestation with a normal birth. Physical examination demonstrated penile hypospadias with the urethral meatus in the posterior third of the shaft. Each testicle had descended into the scrotum. Chromosome analysis using G and Q banding showed a karyotype of 46,X⫹marker. Polymerase chain reaction (PCR) was performed to detect sequences from the SRY and DAZ sequences in peripheral blood as well as provide evidence of SRY but absent DAZ in this case (fig. 1). To confirm that the abnormal chromosome was a Y as well as the presence of SRY but absence of DAZ FISH was done with 4 biotinylated Y chromosome specific DNA probes, including DYZ1 (Yq specific repeat sequence), DYZ3 (Y centromere specific repeat sequence), SRY and DAZ. The probe for DYZ3 and SRY hybridized to the 2 regions of the abnormal chromosome but DYZ1 and DAZ did not hybridize to any region (fig. 2). Based on this evidence we proved that the marker chromosome was a dicentric Y chromosome, and this patient has 2 copies of the SRY sequence in the duplicated short arm and Accepted for publication November 22, 2000.
FIG. 1. Analysis of SRY and DAZ sequences in peripheral blood lymphocytes by PCR amplification. 1, male control. 2, female control. 3, in patient amplified 240 bp SRY fragment was present but 124 bp DAZ fragment was absent.
FIG. 2. Staining by FISH analysis. A, using SRY probe Yp11.3 (red signal), DXZ1 probe X centromere specific repeat sequence (yellow signal) and DYZ1 Yq12 (green signal) demonstrates 2 red, 1 yellow and no green signals. dic, dicentric Y. B, using DAZ probe Yq11.23 and DYZ3 probe (Y centromere specific repeat sequence showed no red and 2 green signals. 1244
MOLECULAR ANALYSIS OF HYPOSPADIAS WITH DICENTRIC Y CHROMOSOME
quence. Individuals who have a dicentric Y with long arm break points are expected to have 2 copies of the SRY sequence in the duplicated short arm.1, 2 FISH was most useful and informative for detecting the SRY sequence and localizing the signal with ease. In fact, it proved that our patient had duplication of the SRY sequence. Tuck-Muller et al reviewed the records of patients with the dicentric Y chromosome and hypospadias, which was noted in 9 of 20 phenotypic males (45%) with long arm break points but not in the 11 in the short arm break point group.2 Our patient had only a long arm break point. Although there is no proof of duplication of the SRY sequence in the patients of Tuck-Muller et al duplication of the SRY sequence may also affect urethral development. Recently 2 candidate genes for azoospermia have been cloned from the azoospermic factor locus on the long arm of the Y chromosome, of which 1 is DAZ. Some infertile men with azoospermia or severe oligospermia have small deletions in regions of the Y chromosome.3 PCR and FISH of the DNA in our patient confirmed absent DAZ. Since there is a high possibility that in our patient future fertility may be impaired because of the absent DAZ sequence, regular longterm followup is necessary.
1245
centromeres of 2 chromosomal segments (different chromosomes or sister chromatids from the same chromosome) fuse, resulting in the loss of genetic material not attached to the centromere. The most common dicentric chromosomes are X and Y. In hypospadias dicentric Y chromosomes have not been widely reported and their unique molecular findings have been studied even less. In the current case of hypospadias the authors used FISH to identify a dicentric Y chromosome with 2 copies of the SRY sequence on the duplicated short arm. In addition, the DAZ gene on the missing long arm segment and associated with male factor infertility is lacking in this child and may have profound implications on his future fertility. Understanding the molecular basis of hypospadias is in its infancy. Case reports such as this should stimulate us to study this ever increasingly common anomaly. In this child a chromosomal rearrangement results in duplication of some genetic material that is involved in gender and perhaps urethral development, and the loss of genetic material that may portend future infertility. The parsimony of this scenario is intriguing but the details of the mechanism leading to this scenario are subject to hypothesis and conjecture, and are not understood to any significant degree. The food for thought and future study is there for the asking and we should seize it so that we expand our knowledge base to beyond the technical. Lane S. Palmer Schneider Children’s Hospital Long Island Jewish Medical Center New Hyde Park, New York
REFERENCES
1. Genuardi, M., Bardoni, B., Floridia, G. et al: Dicentric chromosome Y associated with Leydig cell agenesis and sex reversal. Clin Genet, 47: 38, 1995 2. Tuck-Muller, C. M., Chen, H., Martinez, J. E. et al: Isodicentric Y chromosome: cytogenetic, molecular and clinical studies and review of the literature. Hum Genet, 96: 119, 1995 3. Reijo, R., Lee, T.-Y., Salo, P. et al: Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA-binding protein gene. Nat Genet, 10: 383, 1995 EDITORIAL COMMENT As pediatric urologists, our hypospadias knowledge base continues to grow regarding repair techniques, surgical timing to account for psychological child development and anesthetic considerations. However, the embryological and genetic basis for this anomaly remains rudimentary. The authors present molecular findings in a single case of hypospadias that stimulates those of us interested not only in hypospadias, but also in the molecular basis of congenital anomalies. Dicentric chromosomes are uncommon occurrences in which the
REPLY BY AUTHORS The progress in molecular biology in recent years has helped us to understand several diseases at the gene level, and put to practical use the findings on the mechanism and genetic diagnosis. In pediatric urology as well, several researchers and clinicians have been examining the molecular basis of various disorders, which will enable clinicians to offer more accurate diagnosis and counseling to afflicted patients and their families. Although several investigators have studied the cause of hypospadias, the exact cause remains unknown. Known causes include decreased androgen sensitivity, 5␣-reductase deficiency and chromosomal abnormalities. Since the cause of hypospadias is probably polygenic, it will be difficult to elucidate the mechanism of this disorder completely. We described a boy with hypospadias who had a dicentric Y chromosome. Molecular analysis using PCR and FISH provided evidence of the presence of short arm sequences, including SRY, their duplication and deletion of distal long arm sequences, including DAZ, in our patient. We hope that the molecular findings in this study help to expand our knowledge concerning the cause of hypospadias.