Identification of polymorphisms and balancing selection in the male infertility candidate gene, ornithine decarboxylase antizyme 3

Abstract Background The antizyme family is a group of small proteins that play a role in cell growth and division by regulating the biosynthesis of polyamines (putrescine, spermidine, spermine). Antizymes regulate polyamine levels primarily through binding ornithine decarboxylase (ODC), an enzyme key to polyamine production, and targeting ODC for destruction by the 26S proteosome. Ornithine decarboxylase antizyme 3 (OAZ3) is a testis-specific antizyme paralog and the only antizyme expressed in the mid to late stages of spermatogenesis. Methods To see if mutations in the OAZ3 gene are responsible for some cases of male infertility, we sequenced and evaluated the genomic DNA of 192 infertile men, 48 men of known paternity, and 34 African aborigines from the Mbuti tribe in the Democratic Republic of the Congo. The coding sequence of OAZ3 was further screened for polymorphisms by SSCP analysis in the infertile group and an additional 250 general population controls. Identified polymorphisms in the OAZ3 gene were further subjected to a haplotype analysis using PHASE 2.02 and Arlequin 2.0 software programs. Results A total of 23 polymorphisms were identified in the promoter, exons or intronic regions of OAZ3. The majority of these fell within a region of less than two kilobases. Two of the polymorphisms, -239 A/G in the promoter and 4280 C/T, a missense polymorphism in exon 5, may show evidence of association with male infertility. Haplotype analysis identified 15 different haplotypes, which can be separated into two divergent clusters. Conclusion Mutations in the OAZ3 gene are not a common cause of male infertility. However, the presence of the two divergent haplotypes at high frequencies in all three of our subsamples (infertile, control, African) suggests that they have been maintained in the genome by balancing selection, which was supported by a test of Tajima's D statistic. Evidence for natural selection in this region implies that these haplotypes may be associated with a trait other than infertility. This trait may be related to another function of OAZ3 or a region in tight linkage disequilibrium to the gene.</p

Uniparental Genetic Heritage of Belarusians: Encounter of Rare Middle Eastern Matrilineages with a Central European Mitochondrial DNA Pool

Ethnic Belarusians make up more than 80% of the nine and half million people inhabiting the Republic of Belarus. Belarusians together with Ukrainians and Russians represent the East Slavic linguistic group, largest both in numbers and territory, inhabiting East Europe alongside Baltic-, Finno-Permic- and Turkic-speaking people. Till date, only a limited number of low resolution genetic studies have been performed on this population. Therefore, with the phylogeographic analysis of 565 Y-chromosomes and 267 mitochondrial DNAs from six well covered geographic sub-regions of Belarus we strove to complement the existing genetic profile of eastern Europeans. Our results reveal that around 80% of the paternal Belarusian gene pool is composed of R1a, I2a and N1c Y-chromosome haplogroups – a profile which is very similar to the two other eastern European populations – Ukrainians and Russians. The maternal Belarusian gene pool encompasses a full range of West Eurasian haplogroups and agrees well with the genetic structure of central-east European populations. Our data attest that latitudinal gradients characterize the variation of the uniparentally transmitted gene pools of modern Belarusians. In particular, the Y-chromosome reflects movements of people in central-east Europe, starting probably as early as the beginning of the Holocene. Furthermore, the matrilineal legacy of Belarusians retains two rare mitochondrial DNA haplogroups, N1a3 and N3, whose phylogeographies were explored in detail after de novo sequencing of 20 and 13 complete mitogenomes, respectively, from all over Eurasia. Our phylogeographic analyses reveal that two mitochondrial DNA lineages, N3 and N1a3, both of Middle Eastern origin, might mark distinct events of matrilineal gene flow to Europe: during the mid-Holocene period and around the Pleistocene-Holocene transition, respectively

Novel methylation specific real-time PCR test for the diagnosis of Klinefelter syndrome

The aim of this study was to design a molecular assay for the diagnosis of Klinefelter syndrome (KS), based on the detection of supernumerary X-chromosomes (X-chs). DNA was extracted from peripheral blood samples of twenty-six 47,XXY males; two 46,XY/47,XXY males; twenty-two 46,XY males; and 15 females; and deaminated. Methylation-specific quantitative polymerase chain reaction (MS-qPCR) was performed using primers for unmethylated and methylated copies of the X-ch inactive-specific transcript (XIST-U and XIST-M) gene. X-ch disomy was determined on the basis of XIST methylation status. Degree of mosaicism in the 46,XY/47,XXY males was compared with karyotype and fluorescent in situ hybridization (FISH) results. Data analysis was performed using the Roche® LightCycler software V. 3.5.3., including determination of crossing points (CPs) by fit-point analysis and melting curve analysis. X-ch disomy was detected in all female controls and KS patients; male controls expressed XIST-M only. CPs ranged from 29.5 to 32.5 (standard deviation (s.d.) 0.8) for XIST-U and from 29 to 31 (s.d. 0.6) for XIST-M. Limit of detection of mosaicism was 1%. Based on XIST-U/XIST-M ratios for the two 47,XXY/46,XY patients, the calculated degree of mosaicism (1.8% and 17.8%) was comparable to FISH results (2.3% and 15%, respectively). Turnaround time from DNA deamination to final data analysis was under 9 h. We conclude that MS-qPCR is a sensitive, specific and rapid test for the detection of X-ch disomy, with applicability for the screening and diagnosis of KS, even in the setting of low grade 47,XXY/46,XY mosaicism

Bulbocavernosus muscle area measurement: a novel method to assess androgenic activity

Serum testosterone does not correlate with androgen tissue activity, and it is critical to optimize tools to evaluate such activity in males. Ultrasound measurement of bulbocavernosus muscle (BCM) was used to assess the relationship between the number of CAG repeats (CAGn) in the androgen receptor (AR) and the BCM size; the changes in the number of CAGn over age were also evaluated. Transperineal ultrasound measurement of the BCM was also performed. AR CAGn were determined by high performance liquid chromatography, and morning hormone levels were determined using immunoassays. Forty-eight men had CAG repeat analysis. Twenty-five were 45 years of age, mean 53 years (s.d. = 5.58). The median CAGn was 21 (13-29). BCM area was greater when the number of CAGn were 24 (P = 0.04). There was a linear correlation between the number of CAGn and the BCM area R 2 = 16% (P = 0.01). In the 45 to 65-years-old group, a much stronger negative correlation (R 2 = 29%, P = 0.01) was noticed. In the 19 to 29-years-old group, no such correlation was found (R 2 = 4%, P = 0.36). In older men, the number of CAGn increased with age (R 2 = 32%, P = 0.01). The number of CAGn in the AR correlates with the area of the BCM. Ultrasound assessment of the BCM is an effective surrogate to evaluate end-organ activity of androgens. The number of CAGn may increase with age

Ubiquitin Specific Protease 26 (USP26) expression analysis in human testicular and extragonadal tissues indicates diverse action of USP26 in cell differentiation and tumorigenesis.

Ubiquitin specific protease 26 (USP26), a deubiquitinating enzyme, is highly expressed early during murine spermatogenesis, in round spermatids, and at the blood-testis barrier. USP26 has also been recognized as a regulator of androgen receptor (AR) hormone-induced action involved in spermatogenesis and steroid production in in vitro studies. Prior mutation screening of USP26 demonstrated an association with human male infertility and low testosterone production, but protein localization and expression in the human testis has not been characterized previously. USP26 expression analysis of mRNA and protein was completed using murine and human testis tissue and human tissue arrays. USP26 and AR mRNA levels in human testis were quantitated using multiplex qRT-PCR. Immunofluorescence colocalization studies were performed with formalin-fixed/paraffin-embedded and frozen tissues using primary and secondary antibodies to detect USP26 and AR protein expression. Human microarray dot blots were used to identify protein expression in extra-gonadal tissues. For the first time, expression of USP26 and colocalization of USP26 with androgen receptor in human testis has been confirmed predominantly in Leydig cell nuclei, with less in Leydig cell cytoplasm, spermatogonia, primary spermatocytes, round spermatids, and Sertoli cells. USP26 likely affects regulatory proteins of early spermatogenesis, including androgen receptor with additional activity in round spermatids. This X-linked gene is not testis-specific, with USP26 mRNA and protein expression identified in multiple other human organ tissues (benign and malignant) including androgen-dependent tissues such as breast (myoepithelial cells and secretory luminal cells) and thyroid tissue (follicular cells). USP26/AR expression and interaction in spermatogenesis and androgen-dependent cancer warrants additional study and may prove useful in diagnosis and management of male infertility

USP26 pF1K and pFN2A GST Flexi Vectors.

<p>Adapted from pF1KT7 Flexi Vector map with sequence reference points (Promega: Madison, WI)</p

Mean absolute expression of human USP26 protein in an array of human normal and neoplastic tissues.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098638#pone-0098638-t002" target="_blank">Table 2</a> demonstrates USP26 protein expression in non-testicular tissue including androgen-dependent cancers. Expression ratio is defined as higher expression divided by lower expression. Cancer versus normal column is directional change showing increased or decreased in cancer relative to normal tissue from each organ (+ =  increase as compared to normal tissue; − =  decrease in expression compared with normal tissue). Expression ratio then used to classify expression pattern (Green =  overexpression; White =  ratio +2 to −2 (equivocal expression), Red =  lower expression compared to normal tissue).</p

USP26 and AR protein expression in breast and thyroid tissue by immunofluorescence in formalin-fixed, paraffin-embedded human tissue.

<p><b>4A</b>: USP26 staining in benign breast tissue (paraffin-embedded fixed), mostly cytoplasmic with some nuclear staining in secretory luminal cells (right arrow) and myoepithelial cells (left arrow). <b>4B</b>: USP26 staining in benign thyroid follicular cells (arrows), mostly cytoplasmic localization.</p

USP26 and AR protein expression in normal testis by immunofluorescence.

<p>All images of stained sections were captured at 400x total magnification; scale bar = 100 µm. <b>3A</b>: Colocalization of USP26 and AR in nucleus and cytoplasm of Leydig cell (A), spermatogonia (B), primary spermatocyte (C), and Sertoli cell (D) in normal human testis (frozen tissue). <b>3B</b>: Partial colocalization of USP26 and AR in nucleus and cytoplasm of Sertoli cell (left arrow) and spermatogonia (right arrow) of normal human testis (frozen tissue). <b>3C</b>: Colocalization of USP26 and AR in nucleus and cytoplasm of Sertoli cells (left arrow) and spermatogonia (right arrows) of normal human testis (frozen tissue). <b>3D</b>: USP26 expression in spermatids (double arrows), and USP26/AR partial colocalization in early cells of spermatogenesis (single arrows) of normal human testis (frozen tissue). <b>3E</b>: Negative control demonstrating specificity of antibody without staining of USP26 or AR, or auto-fluorescence of normal human testis (frozen).</p