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Sequence analysis of the X-linked USP26 gene in severe male factor infertility patients and fertile controls
CORRESPONDENCE Sequence analysis of the X-linked USP26 gene in severe male factor infertility patients and fertile controls The 1090C>T,L364F variant of the ubiquitin protease 26 (USP26) gene does not appear to be related to male infertility. Mutations of the USP26 gene do not appear to be a common cause of idiopathic azoospermia or severe oligozoospermia. (Fertil Steril 2008;90:851–2. 2008 by American Society for Reproductive Medicine.)
The ubiquitin protease 26 (USP26) gene was first identified from a screen looking for X-linked genes involved in spermatogenesis (1). Exclusive expression of the gene in the testis was determined in both mice and humans. The specific role of USP26 has not been defined, but deubiquitinating enzymes similar to USP26 play important roles in many cellular processes by reversing the degradation of ubiquitinated proteins (2, 3). Given the testis-specific expression of USP26, its putative role in regulating spermatogenesis, and its location on the X chromosome, which limits the gene to a single allele in an individual, it has been regarded as a likely male infertility candidate gene. Two earlier reports have described polymorphisms identified in the USP26 gene in populations of men with infertility, including men with Sertoli cell only syndrome and maturation arrest. In each case there was an apparent increase in amino acid–changing polymorphisms in the infertile men compared with fertile control subjects (4, 5). In the first report, by Stouffs et al. (4), three changes were identified in the same allele: 370–371insACA, 494T>C, and 1423C>T, which cause the amino acid changes T123–124ins, L165S, and H475Y, respectively (4). These changes were identified in 7.2% of individuals screened with Sertoli cell–only syndrome (SCOS) but in none of the individuals with maturation arrest or with known fertility. These same changes were also identified in the second report, by Paduch et al. (5), at a similar frequency of 9.1% in patients with a known SCOS histology. In addition to these changes, Paduch et al. identified several other variants causing an amino acid change, resulting in a total of Received May 10, 2007; revised June 1, 2007; accepted June 28, 2007. Control samples obtained from the Utah Genetic Reference Project (UGRP) were collected with support from a Public Health Services research grant to the Huntsman General Clinical Research Center, number M01-RR00064, from the National Center for Research Resources. The UGRP was also supported by generous gifts from the W. M. Keck and Delores Dore Eccles Foundations. Reprint requests: Douglas T. Carrell, Ph.D., University of Utah School of Medicine, Andrology and IVF Laboratories, 675 S. Arapeen, #205, Salt Lake City, UT 84108 (FAX: 801-581-6127; E-mail: douglas.carrell@ hsc.utah.edu).
0015-0282/08/$34.00 doi:10.1016/j.fertnstert.2007.06.096
10.6% of patients (20 of 188) having an amino acid change in the gene (5). Only one of these variants, 1090C>T,L364F was found in more than one patient (3.3%). Since the initial reports on variations in the USP26 gene, two additional reports have examined the role of the 370– 371insACA, 494T>C, and 1423C>T haplotype in ethnically defined populations (6, 7). In the report by Ravel et al. (6), the haplotype was identified in significant frequencies in sub-Saharan African, Pygmy, and South and East Asian populations, including those with known fertility. Furthermore, the haplotype was almost absent in other populations tested. Their data strongly suggests that the 370– 371insACA, 494T>C, and 1423C>T haplotype is compatible with male fertility. In the second report examining the haplotype frequency, Stouffs et al. (7) examined a large group of caucasian patients and control subjects and detected the haplotype in a single fertile control, consistent with the findings by Ravel et al. (4, 6). Rather than focus on just the 370–371insACA, 494T>C, and 1423C>T haplotype, the purpose of the present study was to evaluate the full sequence of USP26 to both confirm previously described variants and identify new nucleotide changes in the gene. This was a prospective study in which the DNA from consenting severely oligozoospermic (less than 5 million/mL) or azoospermic patients, collected through the Andrology program at the University of Utah after institutional review board approval, and from fertile control subjects obtained from the Utah Genetic Reference Project (UGRP) at the University of Utah Health Sciences Center was evaluated by direct sequencing for mutations in the USP26 gene. The predominant ethnic background of both groups was Western European (>95%). Individuals previously diagnosed with any condition or treatments connected with infertility (e.g., cystic fibrosis, Klinefelter syndrome, varicocele, chemotherapy, etc.) were not included. In total, 96 men (48 azoospermic, 48 oligozoospermic) were screened for alterations of the USP26 gene. An additional 96 men of known paternity (grandfathers and fathers from the UGRP repository), though semen analysis
Fertility and Sterility Vol. 90, No. 3, September 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
851
parameters were unknown, were also screened as a fertile control group. The six sets of PCR primers described by Paduch et al. were used to amplify the single exon of USP26 (5). All reactions were optimized to give clean ample quantities of DNA. Additional internal primers were designed to allow sequencing in both forward and reverse directions. Sequencing was conducted on an ABI 3700 capillary sequencer (Applied Biosystems, Foster City, CA). Sequence trace files generated by the ABI 3700 were subsequently assembled using Phrap program software and evaluated for alterations using the Phred and Consed sequence analysis programs. In addition to software analysis, sequence trace files were visually examined by a trained technician to specifically identify the presence or absence of previously reported polymorphisms. We identified a total of four variations in USP26 in our study population, as shown in Table 1. Two of the variants, 1609C>G,Q537G and 1549C>T,L517F have not been reported previously. The 1609C>G change was found in a single azoospermic patient, and the 1549C>T variant was found in a single fertile control subject. The 576G>A variant, which does not result in an amino acid change, was identified at frequencies similar to those described previously. Of special interest is the identification of the 1090C>T polymorphism, resulting in the conservative change of leucine to phenylalanine (L364F) in both the infertile patient group and the fertile control subjects at the same frequency (7%). In the original report, the L364F variant was only identified in the patient population. We identified the variant in both patient and control populations at the same frequency, likely owing to a larger control population, indicating that it is compatible with fertility and not a direct cause of infertility. We did not identify the 370–371insACA, 494T>C, and 1423C>T variants, each resulting in an amino acid change, in any of our patients or control subjects. Although these variants were found in both of the original studies, our findings are consistent with those of Ravel et al. and Stouffs et al. that show allele frequencies for these variants are either zero or less than 1% in European and South American populations (6, 7). In conclusion, the present data suggest that mutations in the USP26 gene, and specifically the 1090 C>T,L364F variant, are not a common cause of idiopathic male infertility. The identification of polymorphisms in our study, previously found only in infertile groups, in addition to recent reports on ethnic variation in USP26, further emphasizes
852
Christensen et al.
Correspondence
TABLE 1 Nucleotide changes identified in this study. Frequency Amino Nucleotide acid change change 576G>A 1090C>T 1549C>T 1609C>G
None L364F L517F Q537G
Patients (%)
Control subjects (%)
G(53) A(47) C(93) T(7) C(100) C(99) G(1)
G(47) A(53) C(93) T(7) C(99) T(1) C(100)
Christensen. USP26 gene and male infertility. Fertil Steril 2008.
the need for careful attention to the size and makeup of control groups. Acknowledgments: The authors extend their sincere thanks to all family members who participated in the UGRP and the individuals who consented to DNA collection for the infertile panel.
Greg L. Christensen, Ph.D.a Jeanine Griffina Douglas T. Carrell, Ph.D.a,b,c a Andrology and IVF Laboratories; b Departments of Surgery and Obstetrics and Gynecology; and c Department of Physiology, University of Utah School of Medicine, Salt Lake City, Utah REFERENCES 1. Wang PJ, McCarrey JR, Yang F, Page DC. An abundance of X-linked genes expressed in spermatogonia. Nat Genet 2001;27:422–6. 2. Amerik AY, Hochstrasser M. Mechanism and function of deubiquitinating enzymes. Biochim Biophys Acta 2004;1695:189–207. 3. Nijman SM, Luna-Vargas MP, Velds A, Brummelkamp TR, Dirac AM, Sixma TK, et al. A genomic and functional inventory of deubiquitinating enzymes. Cell 2005;123:773–86. 4. Stouffs K, Lissens W, Tournaye H, Van Steirteghem A, Liebaers I. Possible role of USP26 in patients with severely impaired spermatogenesis. Eur J Hum Genet 2005;13:336–40. 5. Paduch DA, Mielnik A, Schlegel PN. Novel mutations in testis-specific ubiquitin protease 26 gene may cause male infertility and hypogonadism. Reprod Biomed Online 2005;10:747–54. 6. Ravel C, El Houate B, Chantot S, Lourenco D, Dumaine A, Rouba H, et al. Haplotypes, mutations and male fertility: the story of the testis-specific ubiquitin protease USP26. Mol Hum Reprod 2006;12:643–6. 7. Stouffs K, Lissens W, Tournaye H, Van Steirteghem A, Liebaers I. Alterations of the USP26 gene in caucasian men. Int J Androl 2006;29: 614–7.
Vol. 90, No. 3, September 2008
The ubiquitin protease 26 (USP26) gene was first identified from a screen looking for X-linked genes involved in spermatogenesis (1). Exclusive expression of the gene in the testis was determined in both mice and humans. The specific role of USP26 has not been defined, but deubiquitinating enzymes similar to USP26 play important roles in many cellular processes by reversing the degradation of ubiquitinated proteins (2, 3). Given the testis-specific expression of USP26, its putative role in regulating spermatogenesis, and its location on the X chromosome, which limits the gene to a single allele in an individual, it has been regarded as a likely male infertility candidate gene. Two earlier reports have described polymorphisms identified in the USP26 gene in populations of men with infertility, including men with Sertoli cell only syndrome and maturation arrest. In each case there was an apparent increase in amino acid–changing polymorphisms in the infertile men compared with fertile control subjects (4, 5). In the first report, by Stouffs et al. (4), three changes were identified in the same allele: 370–371insACA, 494T>C, and 1423C>T, which cause the amino acid changes T123–124ins, L165S, and H475Y, respectively (4). These changes were identified in 7.2% of individuals screened with Sertoli cell–only syndrome (SCOS) but in none of the individuals with maturation arrest or with known fertility. These same changes were also identified in the second report, by Paduch et al. (5), at a similar frequency of 9.1% in patients with a known SCOS histology. In addition to these changes, Paduch et al. identified several other variants causing an amino acid change, resulting in a total of Received May 10, 2007; revised June 1, 2007; accepted June 28, 2007. Control samples obtained from the Utah Genetic Reference Project (UGRP) were collected with support from a Public Health Services research grant to the Huntsman General Clinical Research Center, number M01-RR00064, from the National Center for Research Resources. The UGRP was also supported by generous gifts from the W. M. Keck and Delores Dore Eccles Foundations. Reprint requests: Douglas T. Carrell, Ph.D., University of Utah School of Medicine, Andrology and IVF Laboratories, 675 S. Arapeen, #205, Salt Lake City, UT 84108 (FAX: 801-581-6127; E-mail: douglas.carrell@ hsc.utah.edu).
0015-0282/08/$34.00 doi:10.1016/j.fertnstert.2007.06.096
10.6% of patients (20 of 188) having an amino acid change in the gene (5). Only one of these variants, 1090C>T,L364F was found in more than one patient (3.3%). Since the initial reports on variations in the USP26 gene, two additional reports have examined the role of the 370– 371insACA, 494T>C, and 1423C>T haplotype in ethnically defined populations (6, 7). In the report by Ravel et al. (6), the haplotype was identified in significant frequencies in sub-Saharan African, Pygmy, and South and East Asian populations, including those with known fertility. Furthermore, the haplotype was almost absent in other populations tested. Their data strongly suggests that the 370– 371insACA, 494T>C, and 1423C>T haplotype is compatible with male fertility. In the second report examining the haplotype frequency, Stouffs et al. (7) examined a large group of caucasian patients and control subjects and detected the haplotype in a single fertile control, consistent with the findings by Ravel et al. (4, 6). Rather than focus on just the 370–371insACA, 494T>C, and 1423C>T haplotype, the purpose of the present study was to evaluate the full sequence of USP26 to both confirm previously described variants and identify new nucleotide changes in the gene. This was a prospective study in which the DNA from consenting severely oligozoospermic (less than 5 million/mL) or azoospermic patients, collected through the Andrology program at the University of Utah after institutional review board approval, and from fertile control subjects obtained from the Utah Genetic Reference Project (UGRP) at the University of Utah Health Sciences Center was evaluated by direct sequencing for mutations in the USP26 gene. The predominant ethnic background of both groups was Western European (>95%). Individuals previously diagnosed with any condition or treatments connected with infertility (e.g., cystic fibrosis, Klinefelter syndrome, varicocele, chemotherapy, etc.) were not included. In total, 96 men (48 azoospermic, 48 oligozoospermic) were screened for alterations of the USP26 gene. An additional 96 men of known paternity (grandfathers and fathers from the UGRP repository), though semen analysis
Fertility and Sterility Vol. 90, No. 3, September 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
851
parameters were unknown, were also screened as a fertile control group. The six sets of PCR primers described by Paduch et al. were used to amplify the single exon of USP26 (5). All reactions were optimized to give clean ample quantities of DNA. Additional internal primers were designed to allow sequencing in both forward and reverse directions. Sequencing was conducted on an ABI 3700 capillary sequencer (Applied Biosystems, Foster City, CA). Sequence trace files generated by the ABI 3700 were subsequently assembled using Phrap program software and evaluated for alterations using the Phred and Consed sequence analysis programs. In addition to software analysis, sequence trace files were visually examined by a trained technician to specifically identify the presence or absence of previously reported polymorphisms. We identified a total of four variations in USP26 in our study population, as shown in Table 1. Two of the variants, 1609C>G,Q537G and 1549C>T,L517F have not been reported previously. The 1609C>G change was found in a single azoospermic patient, and the 1549C>T variant was found in a single fertile control subject. The 576G>A variant, which does not result in an amino acid change, was identified at frequencies similar to those described previously. Of special interest is the identification of the 1090C>T polymorphism, resulting in the conservative change of leucine to phenylalanine (L364F) in both the infertile patient group and the fertile control subjects at the same frequency (7%). In the original report, the L364F variant was only identified in the patient population. We identified the variant in both patient and control populations at the same frequency, likely owing to a larger control population, indicating that it is compatible with fertility and not a direct cause of infertility. We did not identify the 370–371insACA, 494T>C, and 1423C>T variants, each resulting in an amino acid change, in any of our patients or control subjects. Although these variants were found in both of the original studies, our findings are consistent with those of Ravel et al. and Stouffs et al. that show allele frequencies for these variants are either zero or less than 1% in European and South American populations (6, 7). In conclusion, the present data suggest that mutations in the USP26 gene, and specifically the 1090 C>T,L364F variant, are not a common cause of idiopathic male infertility. The identification of polymorphisms in our study, previously found only in infertile groups, in addition to recent reports on ethnic variation in USP26, further emphasizes
852
Christensen et al.
Correspondence
TABLE 1 Nucleotide changes identified in this study. Frequency Amino Nucleotide acid change change 576G>A 1090C>T 1549C>T 1609C>G
None L364F L517F Q537G
Patients (%)
Control subjects (%)
G(53) A(47) C(93) T(7) C(100) C(99) G(1)
G(47) A(53) C(93) T(7) C(99) T(1) C(100)
Christensen. USP26 gene and male infertility. Fertil Steril 2008.
the need for careful attention to the size and makeup of control groups. Acknowledgments: The authors extend their sincere thanks to all family members who participated in the UGRP and the individuals who consented to DNA collection for the infertile panel.
Greg L. Christensen, Ph.D.a Jeanine Griffina Douglas T. Carrell, Ph.D.a,b,c a Andrology and IVF Laboratories; b Departments of Surgery and Obstetrics and Gynecology; and c Department of Physiology, University of Utah School of Medicine, Salt Lake City, Utah REFERENCES 1. Wang PJ, McCarrey JR, Yang F, Page DC. An abundance of X-linked genes expressed in spermatogonia. Nat Genet 2001;27:422–6. 2. Amerik AY, Hochstrasser M. Mechanism and function of deubiquitinating enzymes. Biochim Biophys Acta 2004;1695:189–207. 3. Nijman SM, Luna-Vargas MP, Velds A, Brummelkamp TR, Dirac AM, Sixma TK, et al. A genomic and functional inventory of deubiquitinating enzymes. Cell 2005;123:773–86. 4. Stouffs K, Lissens W, Tournaye H, Van Steirteghem A, Liebaers I. Possible role of USP26 in patients with severely impaired spermatogenesis. Eur J Hum Genet 2005;13:336–40. 5. Paduch DA, Mielnik A, Schlegel PN. Novel mutations in testis-specific ubiquitin protease 26 gene may cause male infertility and hypogonadism. Reprod Biomed Online 2005;10:747–54. 6. Ravel C, El Houate B, Chantot S, Lourenco D, Dumaine A, Rouba H, et al. Haplotypes, mutations and male fertility: the story of the testis-specific ubiquitin protease USP26. Mol Hum Reprod 2006;12:643–6. 7. Stouffs K, Lissens W, Tournaye H, Van Steirteghem A, Liebaers I. Alterations of the USP26 gene in caucasian men. Int J Androl 2006;29: 614–7.
Vol. 90, No. 3, September 2008