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Haplotyping of chromosome 17 in single sperm by multiple displacement DNA amplification
many cases of previously unexplained male infertility, b) it guides diagnostic and treatment decisions, and c) it has potentially serious implications for genetic risk in offspring, given the capability of reproductive technologies. This study sought to define genetic testing acceptance by patients and the incidence of chromosomal abnormalities, Y-chromosome microdeletions, and cystic fibrosis (CF) mutations in this population. DESIGN: A prospective study of genetic testing uptake and findings in infertile men enrolled in a formal genetic counseling and testing program. MATERIALS AND METHODS: Overall, 498 men were evaluated from 1998 –2003 in the Program in the Genetics of Infertility (PROGENI) at the University of California, San Francisco. Indications for evaluation included: oligospermia (n ⫽ 246), non-obstructive azoospermia (n ⫽ 178), congenital absence of the vas deferens (CAVD)/obstructive azoospermia (n ⫽ 59), and other (n ⫽ 16). Genetic counseling and informed consent were performed non-prescriptively prior to testing. Men with oligospermia and non-obstructive azoospermia were offered chromosome analysis (karyotype) and Ychromosome microdeletion analysis (multiplex PCR assay and gel electrophoresis). Men with CAVD or obstructive azoospermia were offered CF mutation analysis and poly-T variant analysis. CF mutations were analyzed by common mutation panel, conformation-sensitive gel electrophoresis (CSGE), or DNA sequence analysis. RESULTS: Overall, 202/246 (82%) men with oligospermia, 140/178 (79%) men with nonobstructive azoospermia, and 46/59 (78%) men with CAVD or obstructive azoospermia opted for genetic testing. In oligospermic men, a chromosomal abnormality was detected in 12/126 (10%) and a Y microdeletion observed in 8/202 (4%). In non-obstructive azoospermic men, a chromosomal abnormality was detected in 12/73 (16%) men and a Y microdeletion in 12/140 (9%) men. In men with CAVD or obstructive azoospermia, 21/46 (46%) were found to carry at least one CF mutation (including 3 compound heterozygotes) and 13/39 (33%) had at least one 5T allele (including 1 homozygote). Detection rates of CF mutations varied widely by mutation method and patient ancestry. CONCLUSION: The incidence of genetic abnormalities in otherwise healthy, infertile men is significant and important. In addition, high rates of patient uptake of genetic testing are observed in a formal, non-prescriptive genetic counseling program. Supported by: None
Wednesday, October 20, 2004 2:30 P.M. O-231 Haplotyping of chromosome 17 in single sperm by multiple displacement DNA amplification. S. Mehri, X.-B. Zhong, A. Borsos, G. Huszar, D. C. Ward, P. Bray-Ward. Yale University School of Medicine, New Haven, CT; University of Debrecen School of Medicine, Debrecen, Hungary. OBJECTIVE: We have adapted the method of haplotyping by MDA (multiple displacement amplification; PNAS, 2002 and 2003) to detect single nucleotide polymorphism in DNA of individual sperm cells, for future application in the study of genetic aspects of male infertility. DESIGN: This study combines three different methods: (a) MDA for whole genome amplification, (b) PCR (polymerase chain reaction) for amplification of specific DNA regions, and (c) single nucleotide polymorphism genotyping with optical thin-film biosensor chips that facilitate the detection of positive hybridization/ligation reactions via color changes at defined regions of the chip surface. MATERIALS AND METHODS: From washed semen samples, groups of 60 –100 sperm were isolated, and from the same sperm suspensions single sperm were also selected with a micromanipulator pipette. The cells were lysed and DNA amplification was carried out directly on the multiple or single cell samples using MDA (Ø29 DNA polymerase, Amersham), generating microgram amounts of DNA with high fidelity locus representation. The MDA amplified DNA was used as a template for PCR. In the present work, we are focusing on chromosome 17; thus we used 20 different chromosome 17 PCR primer pairs. Each individual PCR product was tested using allele-specific single nucleotide polymorphism probes arrayed on the biosensor chips. The target DNA was denatured and incubated on the array in a hybridization/ligation reaction, using a thermostable DNA ligase. All non-ligated molecules were removed by an alkaline wash step. Individual
FERTILITY & STERILITY威
single nucleotide polymorphism were detected as gold to blue/purple color changes on the chip surface, signaling positive ligation events. RESULTS: Sperm from a total of 6 patients were analyzed. The pattern of homozygous and heterozygous alleles for the 20 markers of chromosome 17 were examined by the MDA, PCR and chip analysis of the 60 –100 sperm lysates. For each locus exhibiting heterozygous alleles in the multiple sperm cell lysates, testing of single sperm DNA samples showed one or the other allele, but not both alleles in individual spermatozoa. CONCLUSION: With the adaptation of the MDA technology, we have demonstrated the feasibility of genetic haplotyping in single spermatozoa. The haplotype analysis of specific sperm regions may allow the detection of single nucleotide, or more extensive, genomic changes in the Y and other chromosomes that may be associated with diminished spermatogenesis or sperm function in infertile men. Supported by: Supported by the National Institute of Health
Wednesday, October 20, 2004 2:45 P.M. O-232 Do single nucleotide polymorphisms alter the RNA-binding function of the deleted in azoospermia-like (DAZL) protein? D. C. Sprague, A. R. Ficht, T. J. Kuehl. Texas A&M University System Health Science Center, Temple, TX. OBJECTIVE: To screen DNA extracted from semen samples for single nucleotide polymorphisms (SNPs) in the DAZL gene on chromosome 3 and test whether such mutations have functional effects on RNA-binding of DAZL protein. DESIGN: Anonymous azoospermic (N⫽6), oligozoospermic (N⫽24), and normozoospermic (N⫽2) male partners of infertile couples from a prospectively gathered cohort provided semen residues for SNP analysis. For binding studies, a full-length DAZL cDNA was derived from a commercially available pool of human testicular mRNA. This clone was modified to match two mutations described by Kuo et al, 2002. MATERIALS AND METHODS: DNA was extracted from residues using a modification of a commercially available method. Extracts were screened for SNPs by Polymorphic DNA Technologies, Alameda, CA. Also, a system was devised to investigate the RNA-binding of DAZL made from a wildtype clone and clones with mutations in DAZL exons described by others. For this trial, two mutations, mut1 and mut2, were produced to match the A to G transition in exon 2 and the A to G transition in exon 3 (site of RNA binding domain), respectively. Initial screening for binding was performed using a synthetic RNA shown to bind to murine DAZL and to our clones of human DAZ and DAZL in gel shift assays. First, a DNA probe (CGCGG ATCCT AATAC GACTC ACTAT AGGGG CCACC AACGA CATTG TTTTT TTGTT TTTTT TTTGT TTTTT TTTTG TTTTT TTGTT GATAT AAATA GTGCC CATGG ATCCG CGGGT GTCGG G) was produced. This DNA was gel purified and used for in vitro transcription in the presence of radiolabeled UTP. The labeled RNA was purified and used in the binding reaction. DAZL and its mutants were cloned using the pMAL system (New England Biolabs, Beverly, MA). The fusion proteins of maltose binding protein (MBP), DAZL, and its mutants were expressed and purified onto amylose resin. The binding reaction between RNA and fusion proteins bound to the beads took place at 4 degrees C for 30 minutes. The reaction mixes with beads were applied to small spin columns used in Qiagen’s Oligotex RNA Purification System (Valencia, CA). The flow through was counted in a scintillation counter and data analyzed for retention of labeled RNA. RESULTS: In semen residues from 32 males, 5 sites located in DAZL introns were found to have SNPs. At one site 14 men had a “heterozygous” SNP and 11 men had a “homozygous” SNP. Three other “heterozygous” SNPs were noted in 1, 10, and 5 men. One other “homozygous” SNP was found in 1 man. No SNPs in DAZL exons were found in this cohort and 5/32, including both males with normal semen analyses, had no SNPs. While SNPs in introns may alter slicing of transcripts, our initial screening interest was on SNPs in exons. Labeled RNA was significantly (p⬍0.0002) retained by beads with DAZL proteins from wildtype, mut1, and mut2 compared with MBP beads. However, binding did not differ (P⬎0.52) between the DAZL proteins. CONCLUSION: We have demonstrated SNPs in DAZL introns, but not exons, of a cohort of male factor patients. As this cohort is expanded,
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Wednesday, October 20, 2004 2:30 P.M. O-231 Haplotyping of chromosome 17 in single sperm by multiple displacement DNA amplification. S. Mehri, X.-B. Zhong, A. Borsos, G. Huszar, D. C. Ward, P. Bray-Ward. Yale University School of Medicine, New Haven, CT; University of Debrecen School of Medicine, Debrecen, Hungary. OBJECTIVE: We have adapted the method of haplotyping by MDA (multiple displacement amplification; PNAS, 2002 and 2003) to detect single nucleotide polymorphism in DNA of individual sperm cells, for future application in the study of genetic aspects of male infertility. DESIGN: This study combines three different methods: (a) MDA for whole genome amplification, (b) PCR (polymerase chain reaction) for amplification of specific DNA regions, and (c) single nucleotide polymorphism genotyping with optical thin-film biosensor chips that facilitate the detection of positive hybridization/ligation reactions via color changes at defined regions of the chip surface. MATERIALS AND METHODS: From washed semen samples, groups of 60 –100 sperm were isolated, and from the same sperm suspensions single sperm were also selected with a micromanipulator pipette. The cells were lysed and DNA amplification was carried out directly on the multiple or single cell samples using MDA (Ø29 DNA polymerase, Amersham), generating microgram amounts of DNA with high fidelity locus representation. The MDA amplified DNA was used as a template for PCR. In the present work, we are focusing on chromosome 17; thus we used 20 different chromosome 17 PCR primer pairs. Each individual PCR product was tested using allele-specific single nucleotide polymorphism probes arrayed on the biosensor chips. The target DNA was denatured and incubated on the array in a hybridization/ligation reaction, using a thermostable DNA ligase. All non-ligated molecules were removed by an alkaline wash step. Individual
FERTILITY & STERILITY威
single nucleotide polymorphism were detected as gold to blue/purple color changes on the chip surface, signaling positive ligation events. RESULTS: Sperm from a total of 6 patients were analyzed. The pattern of homozygous and heterozygous alleles for the 20 markers of chromosome 17 were examined by the MDA, PCR and chip analysis of the 60 –100 sperm lysates. For each locus exhibiting heterozygous alleles in the multiple sperm cell lysates, testing of single sperm DNA samples showed one or the other allele, but not both alleles in individual spermatozoa. CONCLUSION: With the adaptation of the MDA technology, we have demonstrated the feasibility of genetic haplotyping in single spermatozoa. The haplotype analysis of specific sperm regions may allow the detection of single nucleotide, or more extensive, genomic changes in the Y and other chromosomes that may be associated with diminished spermatogenesis or sperm function in infertile men. Supported by: Supported by the National Institute of Health
Wednesday, October 20, 2004 2:45 P.M. O-232 Do single nucleotide polymorphisms alter the RNA-binding function of the deleted in azoospermia-like (DAZL) protein? D. C. Sprague, A. R. Ficht, T. J. Kuehl. Texas A&M University System Health Science Center, Temple, TX. OBJECTIVE: To screen DNA extracted from semen samples for single nucleotide polymorphisms (SNPs) in the DAZL gene on chromosome 3 and test whether such mutations have functional effects on RNA-binding of DAZL protein. DESIGN: Anonymous azoospermic (N⫽6), oligozoospermic (N⫽24), and normozoospermic (N⫽2) male partners of infertile couples from a prospectively gathered cohort provided semen residues for SNP analysis. For binding studies, a full-length DAZL cDNA was derived from a commercially available pool of human testicular mRNA. This clone was modified to match two mutations described by Kuo et al, 2002. MATERIALS AND METHODS: DNA was extracted from residues using a modification of a commercially available method. Extracts were screened for SNPs by Polymorphic DNA Technologies, Alameda, CA. Also, a system was devised to investigate the RNA-binding of DAZL made from a wildtype clone and clones with mutations in DAZL exons described by others. For this trial, two mutations, mut1 and mut2, were produced to match the A to G transition in exon 2 and the A to G transition in exon 3 (site of RNA binding domain), respectively. Initial screening for binding was performed using a synthetic RNA shown to bind to murine DAZL and to our clones of human DAZ and DAZL in gel shift assays. First, a DNA probe (CGCGG ATCCT AATAC GACTC ACTAT AGGGG CCACC AACGA CATTG TTTTT TTGTT TTTTT TTTGT TTTTT TTTTG TTTTT TTGTT GATAT AAATA GTGCC CATGG ATCCG CGGGT GTCGG G) was produced. This DNA was gel purified and used for in vitro transcription in the presence of radiolabeled UTP. The labeled RNA was purified and used in the binding reaction. DAZL and its mutants were cloned using the pMAL system (New England Biolabs, Beverly, MA). The fusion proteins of maltose binding protein (MBP), DAZL, and its mutants were expressed and purified onto amylose resin. The binding reaction between RNA and fusion proteins bound to the beads took place at 4 degrees C for 30 minutes. The reaction mixes with beads were applied to small spin columns used in Qiagen’s Oligotex RNA Purification System (Valencia, CA). The flow through was counted in a scintillation counter and data analyzed for retention of labeled RNA. RESULTS: In semen residues from 32 males, 5 sites located in DAZL introns were found to have SNPs. At one site 14 men had a “heterozygous” SNP and 11 men had a “homozygous” SNP. Three other “heterozygous” SNPs were noted in 1, 10, and 5 men. One other “homozygous” SNP was found in 1 man. No SNPs in DAZL exons were found in this cohort and 5/32, including both males with normal semen analyses, had no SNPs. While SNPs in introns may alter slicing of transcripts, our initial screening interest was on SNPs in exons. Labeled RNA was significantly (p⬍0.0002) retained by beads with DAZL proteins from wildtype, mut1, and mut2 compared with MBP beads. However, binding did not differ (P⬎0.52) between the DAZL proteins. CONCLUSION: We have demonstrated SNPs in DAZL introns, but not exons, of a cohort of male factor patients. As this cohort is expanded,
S93