A multi-exon deletion within WWOX is associated with a 46,XY disorder of sex development

Disorders of sex development (DSD) are congenital conditions where chromosomal, gonad or genital development is atypical. In a significant proportion of 46,XY DSD cases it is not possible to identify a causative mutation, making genetic counseling difficult and potentially hindering optimal treatment. Here, we describe the analysis of a 46,XY DSD patient that presented at birth with ambiguous genitalia. Histological analysis of the surgically removed gonads showed bilateral undifferentiated gonadal tissue and immature testis, both containing malignant germ cells. We screened genomic DNA from this patient for deletions and duplications using an Illumina whole-genome SNP microarray. This analysis revealed a heterozygous deletion within the WWOX gene on chromosome 16, removing exons 6-8. Analysis of parental DNA showed that the deletion was inherited from the mother. cDNA analysis confirmed that the deletion maintained the reading frame, with exon 5 being spliced directly onto exon 9. This deletion is the first description of a germline rearrangement affecting the coding sequence of WWOX in humans. Previously described Wwox knockout mouse models showed gonadal abnormalities, supporting a role for WWOX in human gonad development

Sex reversal following deletion of a single distal enhancer of Sox9

Cell fate decisions require appropriate regulation of key genes. Sox9, a direct target of SRY, is pivotal in mammalian sex determination. In vivo high-throughput chromatin accessibility techniques, transgenic assays, and genome editing revealed several novel gonadal regulatory elements in the 2-megabase gene desert upstream of Sox9. Although others are redundant, enhancer 13 (Enh13), a 557–base pair element located 565 kilobases 5′ from the transcriptional start site, is essential to initiate mouse testis development; its deletion results in XY females with Sox9 transcript levels equivalent to those in XX gonads. Our data are consistent with the time-sensitive activity of SRY and indicate a strict order of enhancer usage. Enh13 is conserved and embedded within a 32.5-kilobase region whose deletion in humans is associated with XY sex reversal, suggesting that it is also critical in humans

Global Mapping of DNA Methylation in Mouse Promoters Reveals Epigenetic Reprogramming of Pluripotency Genes

DNA methylation patterns are reprogrammed in primordial germ cells and in preimplantation embryos by demethylation and subsequent de novo methylation. It has been suggested that epigenetic reprogramming may be necessary for the embryonic genome to return to a pluripotent state. We have carried out a genome-wide promoter analysis of DNA methylation in mouse embryonic stem (ES) cells, embryonic germ (EG) cells, sperm, trophoblast stem (TS) cells, and primary embryonic fibroblasts (pMEFs). Global clustering analysis shows that methylation patterns of ES cells, EG cells, and sperm are surprisingly similar, suggesting that while the sperm is a highly specialized cell type, its promoter epigenome is already largely reprogrammed and resembles a pluripotent state. Comparisons between pluripotent tissues and pMEFs reveal that a number of pluripotency related genes, including Nanog, Lefty1 and Tdgf1, as well as the nucleosome remodeller Smarcd1, are hypomethylated in stem cells and hypermethylated in differentiated cells. Differences in promoter methylation are associated with significant differences in transcription levels in more than 60% of genes analysed. Our comparative approach to promoter methylation thus identifies gene candidates for the regulation of pluripotency and epigenetic reprogramming. While the sperm genome is, overall, similarly methylated to that of ES and EG cells, there are some key exceptions, including Nanog and Lefty1, that are highly methylated in sperm. Nanog promoter methylation is erased by active and passive demethylation after fertilisation before expression commences in the morula. In ES cells the normally active Nanog promoter is silenced when targeted by de novo methylation. Our study suggests that reprogramming of promoter methylation is one of the key determinants of the epigenetic regulation of pluripotency genes. Epigenetic reprogramming in the germline prior to fertilisation and the reprogramming of key pluripotency genes in the early embryo is thus crucial for transmission of pluripotency

Defending the genome from the enemy within:mechanisms of retrotransposon suppression in the mouse germline

The viability of any species requires that the genome is kept stable as it is transmitted from generation to generation by the germ cells. One of the challenges to transgenerational genome stability is the potential mutagenic activity of transposable genetic elements, particularly retrotransposons. There are many different types of retrotransposon in mammalian genomes, and these target different points in germline development to amplify and integrate into new genomic locations. Germ cells, and their pluripotent developmental precursors, have evolved a variety of genome defence mechanisms that suppress retrotransposon activity and maintain genome stability across the generations. Here, we review recent advances in understanding how retrotransposon activity is suppressed in the mammalian germline, how genes involved in germline genome defence mechanisms are regulated, and the consequences of mutating these genome defence genes for the developing germline

Synergistic Effect of SRY and Its Direct Target, WDR5, on Sox9 Expression

SRY is a sex-determining gene that encodes a transcription factor, which triggers male development in most mammals. The molecular mechanism of SRY action in testis determination is, however, poorly understood. In this study, we demonstrate that WDR5, which encodes a WD-40 repeat protein, is a direct target of SRY. EMSA experiments and ChIP assays showed that SRY could bind to the WDR5 gene promoter directly. Overexpression of SRY in LNCaP cells significantly increased WDR5 expression concurrent with histone H3K4 methylation on the WDR5 promoter. To specifically address whether SRY contributes to WDR5 regulation, we introduced a 4-hydroxy-tamoxifen-inducible SRY allele into LNCaP cells. Conditional SRY expression triggered enrichment of SRY on the WDR5 promoter resulting in induction of WDR5 transcription. We found that WDR5 was self regulating through a positive feedback loop. WDR5 and SRY interacted and were colocalized in cells. In addition, the interaction of WDR5 with SRY resulted in activation of Sox9 while repressing the expression of β-catenin. These results suggest that, in conjunction with SRY, WDR5 plays an important role in sex determination

Downregulation of microRNA-383 is associated with male infertility and promotes testicular embryonal carcinoma cell proliferation by targeting IRF1

Our previous studies have shown that microRNA-383 (miR-383) expression is downregulated in the testes of infertile men with maturation arrest (MA). However, the underlying mechanisms of miR-383 involved in the pathogenesis of MA remain unknown. In this study, we showed that downregulation of miR-383 was associated with hyperactive proliferation of germ cells in patients with mixed patterns of MA. Overexpression of miR-383 in NT2 (testicular embryonal carcinoma) cells resulted in suppression of proliferation, G1-phase arrest and induction of apoptosis, whereas silencing of miR-383 reversed these effects. The effects of miR-383 were mediated through targeting a tumor suppressor, interferon regulatory factor-1 (IRF1), and miR-383 was negatively correlated with IRF1 protein expression in vivo. miR-383 inhibited IRF1 by affecting its mRNA stability, which subsequently reduced the levels of the targets of IRF1, namely cyclin D1, CDK2 and p21. Downregulation of IRF1 or cyclin D1, but not that of CDK2, enhanced miR-383-mediated effects, whereas silencing of p21 partially inhibited the effects of miR-383. Moreover, miR-383 downregulated CDK4 by increasing proteasome-dependent degradation of CDK4, which in turn resulted in an inhibition of phosphorylated retinoblastoma protein (pRb) phosphorylation. These results suggest that miR-383 functions as a negative regulator of proliferation by targeting IRF1, in part, through inactivation of the pRb pathway. Abnormal testicular miR-383 expression may potentiate the connections between male infertility and testicular germ cell tumor

Dicer1 Depletion in Male Germ Cells Leads to Infertility Due to Cumulative Meiotic and Spermiogenic Defects

Background: Spermatogenesis is a complex biological process that requires a highly specialized control of gene expression. In the past decade, small non-coding RNAs have emerged as critical regulators of gene expression both at the transcriptional and post-transcriptional level. DICER1, an RNAse III endonuclease, is essential for the biogenesis of several classes of small RNAs, including microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), but is also critical for the degradation of toxic transposable elements. In this study, we investigated to which extent DICER1 is required for germ cell development and the progress of spermatogenesis in mice.Principal Findings: We show that the selective ablation of Dicer1 at the early onset of male germ cell development leads to infertility, due to multiple cumulative defects at the meiotic and post-meiotic stages culminating with the absence of functional spermatozoa. Alterations were observed in the first spermatogenic wave and include delayed progression of spermatocytes to prophase I and increased apoptosis, resulting in a reduced number of round spermatids. The transition from round to mature spermatozoa was also severely affected, since the few spermatozoa formed in mutant animals were immobile and misshapen, exhibiting morphological defects of the head and flagellum. We also found evidence that the expression of transposable elements of the SINE family is up-regulated in Dicer1-depleted spermatocytes.Conclusions/Significance: Our findings indicate that DICER1 is dispensable for spermatogonial stem cell renewal and mitotic proliferation, but is required for germ cell differentiation through the meiotic and haploid phases of spermatogenesis

Identification of De Novo Copy Number Variants Associated with Human Disorders of Sexual Development

Disorders of sexual development (DSD), ranging in severity from genital abnormalities to complete sex reversal, are among the most common human birth defects with incidence rates reaching almost 3%. Although causative alterations in key genes controlling gonad development have been identified, the majority of DSD cases remain unexplained. To improve the diagnosis, we screened 116 children born with idiopathic DSD using a clinically validated array-based comparative genomic hybridization platform. 8951 controls without urogenital defects were used to compare with our cohort of affected patients. Clinically relevant imbalances were found in 21.5% of the analyzed patients. Most anomalies (74.2%) evaded detection by the routinely ordered karyotype and were scattered across the genome in gene-enriched subtelomeric loci. Among these defects, confirmed de novo duplication and deletion events were noted on 1p36.33, 9p24.3 and 19q12-q13.11 for ambiguous genitalia, 10p14 and Xq28 for cryptorchidism and 12p13 and 16p11.2 for hypospadias. These variants were significantly associated with genitourinary defects (P = 6.08×10−12). The causality of defects observed in 5p15.3, 9p24.3, 22q12.1 and Xq28 was supported by the presence of overlapping chromosomal rearrangements in several unrelated patients. In addition to known gonad determining genes including SRY and DMRT1, novel candidate genes such as FGFR2, KANK1, ADCY2 and ZEB2 were encompassed. The identification of risk germline rearrangements for urogenital birth defects may impact diagnosis and genetic counseling and contribute to the elucidation of the molecular mechanisms underlying the pathogenesis of human sexual development

Human RSPO1/R-spondin1 Is Expressed during Early Ovary Development and Augments β-Catenin Signaling

Human testis development starts from around 42 days post conception with a transient wave of SRY expression followed by up-regulation of testis specific genes and a distinct set of morphological, paracrine and endocrine events. Although anatomical changes in the ovary are less marked, a distinct sub-set of ovary specific genes are also expressed during this time. The furin-domain containing peptide R-spondin1 (RSPO1) has recently emerged as an important regulator of ovary development through up-regulation of the WNT/β-catenin pathway to oppose testis formation. Here, we show that RSPO1 is upregulated in the ovary but not in the testis during critical early stages of gonad development in humans (between 6–9 weeks post conception), whereas the expression of the related genes WNT4 and CTNNB1 (encoding β catenin) is not significantly different between these tissues. Furthermore, reduced R-spondin1 function in the ovotestis of an individual (46,XX) with a RSPO1 mutation leads to reduced β-catenin protein and WNT4 mRNA levels, consistent with down regulation of ovarian pathways. Transfection of wild-type RSPO1 cDNA resulted in weak dose-dependent activation of a β-catenin responsive TOPFLASH reporter (1.8 fold maximum), whereas co-transfection of CTNNB1 (encoding β-catenin) with RSPO1 resulted in dose-dependent synergistic augmentation of this reporter (approximately 10 fold). Furthermore, R-spondin1 showed strong nuclear localization in several different cell lines. Taken together, these data show that R-spondin1 is upregulated during critical stages of early human ovary development and may function as a tissue-specific amplifier of β-catenin signaling to oppose testis determination