Mol. Hum. Reprod. Advance Access originally published online on October 14, 2005
Molecular Human Reproduction 2005 11(9):673-675; doi:10.1093/molehr/gah232
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Mutations in the chromosome pairing gene FKBP6 are not a common cause of non-obstructive azoospermia
1Department of Obstetrics and Gynecology, Center for Reproductive Medicine and 2Department of Clinical Genetics, Academic Medical Center, Amsterdam, The Netherlands
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Academic Medical Center, Meibergdreef 9, H4-205, 1105 AZ Amsterdam, The Netherlands. E-mail: g.h.westerveld{at}amc.uva.nl
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Abstract |
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Although it is generally thought that spermatogenic failure has a genetic background, to date only a limited percentage of men with non-obstructive azoospermia (NOA) are diagnosed with a genetic defect. The only common and well-established genetic causes of NOA in humans are numerical and structural chromosomal abnormalities and Y-chromosome deletions. In addition, some infrequent mutations have been identified in the ubiquitin-specific protease 9, Y-linked (USP9Y) and the synaptonemal complex protein 3 (SYCP3) gene that cause azoospermia. FK506-binding protein 6 (Fkbp6) is a newly discovered component of the synaptonemal complex (SC), which is essential for proper chromosome pairing and meiotic division. A null mutation of the Fkbp6 gene causes azoospermia in mice as well as in rats. We tested the hypothesis whether mutations in this gene can also cause azoospermia in humans. We performed a mutation screen in 51 men with NOA through direct sequencing methods. No homozygous mutations were identified. Two heterozygous mutations (T173T and R183C) were identified, which are likely to disrupt FKBP6 protein function. However, both mutations were also found in a group of 218 normospermic controls indicating that one FKBP6 allele appears to be sufficient for normal spermatogenesis. In conclusion, our results suggest that genetic defects in FKBP6 can be excluded as a common cause of azoospermia in humans.
Key words: azoospermia/FKBP6/genetics/meiosis/spermatogenic failure
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Introduction |
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Currently, approximately 20% of men with non-obstructive azoospermia (NOA) are diagnosed with an infertility causing genetic defect, i.e. a karyotype abnormality or a Y-chromosome deletion (Fedder et al., 2004
; Gianotten et al., 2004; Repping et al., 2004). In addition, some infrequent mutations have been identified in the ubiquitin-specific protease 9, Y-linked (USP9Y) and the synaptonemal complex protein 3 (SYCP3) gene that cause azoospermia (Sun et al., 1999; Miyamoto et al., 2003). It is thought, however, that a substantial part of the remaining unexplained cases also have a genetic aetiology.
One of the genes that may play a role in NOA is FK506-binding protein 6 (FKBP6). FKBP6 is located in chromosomal region 7q11.23 and is a member of the immunophilins FKBP family of which all members contain a prolyl isomerase/FK506-binding domain (FKBP_C) and a tetraticopeptide protein–protein interaction domain (TRP). Northern blot analysis has shown that FKBP6 is predominantly expressed in testis tissue (Meng et al., 1998).
Recently, an article was published showing that Fkbp6 in mice is essential for male fertility (Crackower et al., 2003). The Fkbp6 protein is a component of the synaptonemal complex (SC) (Crackower et al., 2003), a structure required for proper homologous recombination during prophase of the first meiotic division (Heyting, 1996; Champion and Hawley, 2002). Knockout male mice lacking exon 6 and 7 of the Fkbp6 gene are completely sterile because of a block in meiosis and cell death of meiotic spermatocytes but are otherwise healthy. Mouse Fkbp6 mRNA expression is restricted to testis. A spontaneous deletion of exon 8 in the Fkbp6 gene of the ‘aspermia’ (as/as) rat also results in complete sterility (Crackower et al., 2003). The testicular phenotype of this as/as rat closely resembles that of the Fkbp6–/– mutant mouse (Ikadai et al., 1992; Noguchi et al., 1999, 2004). Both the heterozygous Fbp6+/– mouse and the as rat are fertile (Noguchi et al., 1999; Crackower et al., 2003). Fertility and meiosis are also normal in Fkbp6 mutant females.
Taken together, these studies suggest that FKBP6 is an excellent candidate gene for human spermatogenic failure, and thus we sought to determine whether mutations in FKBP6 can cause NOA in humans.
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Materials and methods |
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As part of our ongoing research regarding the genetics of spermatogenic failure, we consecutively included all men that attended our outpatient clinic and gave informed consent from January 1998 to December 2004. Men with a history of orchitis, surgery of the vasa deferentia, bilateral orchidectomy, chemo- or radiotherapy, obstructive azoospermia, bilateral cryptorchidism, numerical or structural chromosomal abnormalities, and Y-chromosome deletions were excluded. Men with idiopathic NOA were included as patients in this study. Normospermic men, defined as having a total sperm count of more than 40 x 106 and normal motility and morphology in two semen samples, were included as controls. Semen analyses were performed, according to WHO guidelines (World Health Organization, 1999
). The Institutional Review Board of the Academic Medical Center approved this study. DNA from all men was extracted from peripheral blood leucocytes, according to standard procedures.
We amplified the entire coding sequence including the intron/exon boundaries of FKBP6. Seven pairs of primers were designed with the use of Primer3 (Table I) (Rozen and Skaletsky, 2000), using NM_003602
[GenBank]
as reference sequence. PCR was carried out in a total volume of 20 µl and contained 25 ng DNA, 1 M betaine (Sigma, St. Louise, MO, USA), 4.0 µl 1 x betaine buffer (0.25 M Tris [pH 9.2], 70 mM (NH4)2SO4, 10% dimethylsulphoxide [DMSO] and 1.5% Tween), 0.2 mM dNTPs, 10 pmol forward and reverse primer, 3.75 mM MgCl2 and 0.5 U Taq polymerase. We used a touchdown PCR program with a temperature range of 66–54°C with a 2°C decrement per cycle and 1 cycle increment per temperature step and a final amplification for 20 cycles at 94°C for 30 s, 54°C for 30 s and 72°C for 30 s with a final extension at 72°C for 5 min.
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Mutation screening was performed through direct sequencing of both sense and antisense strands, using the same primers as those used for PCR, on an automated ABI Prism 3100 Genetic Analyzer (Applied Biosystems Foster City, CA, USA). Sequence analyses were performed with the use of the CodonCode Aligner software (CodonCode Corporation, Dedham, MA, USA).
We applied the ESEfinder (Exonic Splicing Enhancer) program (http://rulai.cshl.edu/tools/ESE/) to determine the effect of apparently silent, exonic variants on splicing activity. Similarly, to evaluate the effect of intronic variants on the branch site sequence, thereby affecting splicing activity, we used the Alex Dong Li’s SpliceSiteFinder (http://www.genet.sickkids.on.ca/~ali/splicesitefinder.html). Each silent mutation and intronic variant that was predicted to alter splice site activity or branch site sequence was evaluated in our control group of normospermic men through direct sequencing.
We applied the BLOSUM62 Substitution Scoring Matrix, this matrix is designed by Henikoff and Henikoff (see reference) and not by a company, to describe the putative impact of identified amino acid changes (Henikoff and Henikoff, 1992). The BLOSUM62 matrix is a matrix in which every possible amino acid identity and substitution is assigned a score based on the observed frequencies of such occurrences in alignments of related proteins. Frequently observed substitutions receive positive scores (highest score: 11), and seldom observed substitutions are given negative scores (lowest score: –4).
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Results |
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We included 51 men with NOA as patients and 218 men with normozoospermia as controls in this study.
Screening for mutations in the FKBP6 gene of the azoospermic men revealed no homozygous mutations. Four heterozygous single-nucleotide changes were found. One of these four variants was a missense mutation (R183C), two were silent mutations (T173T and L198L) and one was an intronic variant (IVS8–36G). All these variants were found only once, except for the L198L mutation, which occurred twice in the patient population.
The R183C missense mutation was a C
T transition at the first position of codon 183 that changes an arginine into a cysteine. This amino acid change is considered to be significant, because it gives a –3 score in the BLOSUM62 matrix. In addition, it is located in the functional TRP domain.
Analysis of the silent and intronic mutations showed that the T173T mutation is predicted to remove the ESE consensus motifs of two SR (serine/arginine rich) proteins, namely SC35 and SRp40, and thereby potentially affects correct splicing. This T137T mutation is located in the functional FKBP_C domain. The L198L and the IVS8–36G were not predicted to alter splice site activity and thus were not further investigated.
We then screened the normospermic controls for the presence of the R183C and the T173T mutation. The R183C mutation was found in one normospermic man, and the T173T mutation was found in three normospermic men.
The patient with the R183C mutation inherited this mutation from his father, who fathered two sons spontaneously. No family members were available to study the inheritance pattern of the man with the T173T mutation.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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To date, research on the genetic aetiology of NOA has been relatively unrewarding, despite the overwhelming number of well-described candidate genes in the last decade (Matzuk and Lamb, 2002
).
A mutation screen of FKBP6 in 51 azoospermic men revealed no homozygous mutations in FKBP6. Four heterozygous single-nucleotide changes were found. Two of four, namely T173T and R183C, were predicted to result in a disrupted protein. However, both mutations were also found in normospermic controls, and in one case the mutation was paternally transmitted. These data suggest that one allele of FKBP6 is sufficient to preserve normal spermatogenesis. Furthermore, the R183C mutation does not appear to have a dominant negative effect on the function of the wild-type allele, such as previously shown for the heterozygous 643delA mutation in the SYCP3 gene (Miyamoto et al., 2003).
FKBP6 is located in a region that is deleted in patients with Williams–Beuren syndrome, which is an autosomal dominant congenital developmental disorder (Meng et al., 1998; Peoples et al., 2000). Interestingly, a recent study described a man with William–Beuren syndrome, who spontaneously fathered a son (Metcalfe et al., 2005). This case report and our current results suggest that heterozygous mutations as well as heterozygous deletions do not cause azoospermia.
In conclusion, heterozygous mutations indeed occur in FKBP6, but they occur infrequently and do not cause NOA. Although homozygous mutations might cause NOA, they were not detected in our patient population. Thus, genetic defects in FKBP6 appear not to be a common cause of NOA in humans.
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References |
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Submitted on July 21, 2005; revised on September 12, 2005; accepted on September 21, 2005
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