Double Proton Tautomerism via Intra- or Intermolecular Pathways? The Case of Tetramethyl Reductic Acid Studied by Dynamic NMR: Hydrogen Bond Association, Solvent and Kinetic H/D Isotope Effects.

Using dynamic liquid-state NMR spectroscopy a degenerate double proton tautomerism was detected in tetramethyl reductic acid (TMRA) dissolved in toluene-d8 and in CD2Cl2. Similar to vitamin C, TMRA belongs to the class of reductones of biologically important compounds. The tautomerism involves an intramolecular HH transfer that interconverts the peripheric and the central positions of the two OH groups. It is slow in the NMR time scale around 200 K and fast at room temperature. Pseudo-first-order rate constants of the HH transfer and of the HD transfer after suitable deuteration were obtained by line shape analyses. Interestingly, the chemical shifts were found to be temperature dependent carrying information about an equilibrium between a hydrogen bonded dimer and a monomer forming two weak intramolecular hydrogen bonds. The structures of the monomer and the dimer are discussed. The latter may consist of several rapidly interconverting hydrogen-bonded associates. A way was found to obtain the enthalpies and entropies of dissociation, which allowed us to convert the pseudo-first-order rate constants of the reaction mixture into first-order rate constants of the tautomerization of the monomer. Surprisingly, these intrinsic rate constants were the same for toluene-d8 and CD2Cl2, but in the latter solvent more monomer is formed. This finding is attributed to the dipole moment of the TMRA monomer, compensated in the dimer, and to the larger dielectric constant of CD2Cl2. Within the margin of error, the kinetic HH/HD isotope effects were found to be of the order of 3 but independent of temperature. That finding indicates a stepwise HH transfer involving a tunnel mechanism along a double barrier pathway. The Arrhenius curves were described in terms of the Bell-Limbach tunneling model.

Four Novel NR5A1 Mutations in 46,XY Gonadal Dysgenesis Patients Including Frameshift Mutations with Altered Subcellular SF-1 Localization.

46,XY gonadal dysgenesis (46,XY GD) is a disorder of sexual development caused by mutations in genes involved in early gonadal development (bipotential gonads) and testis differentiation. In 46,XY GD individuals, mutations of the SRY gene are detected most frequently, followed by mutations in the NR5A1 (SF-1) gene, but in a lot of cases, the underlying molecular mechanism remains elusive. In this study, we retrospectively performed sequence analyses of the NR5A1 (SF-1) gene in 84 patients with complete, partial, and syndromic forms of 46,XY GD. In total, 7 heterozygous mutations were found in 6 of 84 patients (7.1%). Among these, we identified 4 mutations that, to the best of our knowledge, have not been reported before (c.268G>T, c.369del, c.871-1G>C, and c.893T>C). Transfection of different mutations revealed altered subcellular localization of the mutant SF-1 protein in the case of the frameshift mutations, indicating an impaired protein function. In conclusion, we present 4 novel mutations of the NR5A1 gene associated with 46,XY GD together with in vitro data pointing towards a possible functional impairment of the mutant SF-1 proteins.

A mutation creating an upstream initiation codon in the SOX9 5' UTR causes acampomelic campomelic dysplasia.

BACKGROUND: Campomelic dysplasia (CD) is a semilethal developmental disorder caused by mutations in and around SOX9. CD is characterized by multiple skeletal malformations including bending (campomelia) of long bones. Surviving patients frequently have the acampomelic form of CD (ACD). METHODS: This is a single case report on a patient with clinical and radiological features of ACD who has no mutation in the SOX9 protein-coding sequence nor a translocation with breakpoint in the SOX9 regulatory domain. We include functional studies of the novel mutant protein in vitro and in cultured cells. RESULTS: The patient was found to have a de novo heterozygous mutation c.-185G>A in the SOX9 5'UTR. The mutation creates an upstream translation start codon, uAUG, with a much better fit of its flanking sequence to the Kozak consensus than the wild-type AUG. By in vitro transcription-translation and transient transfection into COS-7 cells, we show that the uAUG leads to translation of a short peptide from a reading frame that terminates just after the wild-type AUG start codon. This results in reduced translation of the wild-type protein, compatible with the milder phenotype of the patient. CONCLUSION: Findings support the notion that more mildly affected, surviving CD/ACD patients carry mutant SOX9 alleles with residual expression of SOX9 wild-type protein. Although rarely described in human genetic disease and for the first time here for CD, mutations creating upstream AUG codons may be more common than generally assumed.

Sox9 and Sox8 protect the adult testis from male-to-female genetic reprogramming and complete degeneration.

The new concept of mammalian sex maintenance establishes that particular key genes must remain active in the differentiated gonads to avoid genetic sex reprogramming, as described in adult ovaries after Foxl2 ablation. Dmrt1 plays a similar role in postnatal testes, but the mechanism of adult testis maintenance remains mostly unknown. Sox9 and Sox8 are required for postnatal male fertility, but their role in the adult testis has not been investigated. Here we show that after ablation of Sox9 in Sertoli cells of adult, fertile Sox8(-/-) mice, testis-to-ovary genetic reprogramming occurs and Sertoli cells transdifferentiate into granulosa-like cells. The process of testis regression culminates in complete degeneration of the seminiferous tubules, which become acellular, empty spaces among the extant Leydig cells. DMRT1 protein only remains in non-mutant cells, showing that SOX9/8 maintain Dmrt1 expression in the adult testis. Also, Sox9/8 warrant testis integrity by controlling the expression of structural proteins and protecting Sertoli cells from early apoptosis. Concluding, this study shows that, in addition to its crucial role in testis development, Sox9, together with Sox8 and coordinately with Dmrt1, also controls adult testis maintenance.

FGFR2 mutation in 46,XY sex reversal with craniosynostosis.

Patients with 46,XY gonadal dysgenesis (GD) exhibit genital anomalies, which range from hypospadias to complete male-to-female sex reversal. However, a molecular diagnosis is made in only 30% of cases. Heterozygous mutations in the human FGFR2 gene cause various craniosynostosis syndromes including Crouzon and Pfeiffer, but testicular defects were not reported. Here, we describe a patient whose features we would suggest represent a new FGFR2-related syndrome, craniosynostosis with XY male-to-female sex reversal or CSR. The craniosynostosis patient was chromosomally XY, but presented as a phenotypic female due to complete GD. DNA sequencing identified the FGFR2c heterozygous missense mutation, c.1025G>C (p.Cys342Ser). Substitution of Cys342 by Ser or other amino acids (Arg/Phe/Try/Tyr) has been previously reported in Crouzon and Pfeiffer syndrome. We show that the 'knock-in' Crouzon mouse model Fgfr2c(C342Y/C342Y) carrying a Cys342Tyr substitution displays XY gonadal sex reversal with variable expressivity. We also show that despite FGFR2c-Cys342Tyr being widely considered a gain-of-function mutation, Cys342Tyr substitution in the gonad leads to loss of function, as demonstrated by sex reversal in Fgfr2c(C342Y/-) mice carrying the knock-in allele on a null background. The rarity of our patient suggests the influence of modifier genes which exacerbated the testicular phenotype. Indeed, patient whole exome analysis revealed several potential modifiers expressed in Sertoli cells at the time of testis determination in mice. In summary, this study identifies the first FGFR2 mutation in a 46,XY GD patient. We conclude that, in certain rare genetic contexts, maintaining normal levels of FGFR2 signaling is important for human testis determination.

Copy number variation of two separate regulatory regions upstream of SOX9 causes isolated 46,XY or 46,XX disorder of sex development.

BACKGROUND: SOX9 mutations cause the skeletal malformation syndrome campomelic dysplasia in combination with XY sex reversal. Studies in mice indicate that SOX9 acts as a testis-inducing transcription factor downstream of SRY, triggering Sertoli cell and testis differentiation. An SRY-dependent testis-specific enhancer for Sox9 has been identified only in mice. A previous study has implicated copy number variations (CNVs) of a 78 kb region 517-595 kb upstream of SOX9 in the aetiology of both 46,XY and 46,XX disorders of sex development (DSD). We wanted to better define this region for both disorders. RESULTS: By CNV analysis, we identified SOX9 upstream duplications in three cases of SRY-negative 46,XX DSD, which together with previously reported duplications define a 68 kb region, 516-584 kb upstream of SOX9, designated XXSR (XX sex reversal region). More importantly, we identified heterozygous deletions in four families with SRY-positive 46,XY DSD without skeletal phenotype, which define a 32.5 kb interval 607.1-639.6 kb upstream of SOX9, designated XY sex reversal region (XYSR). To localise the suspected testis-specific enhancer, XYSR subfragments were tested in cell transfection and transgenic experiments. While transgenic experiments remained inconclusive, a 1.9 kb SRY-responsive subfragment drove expression specifically in Sertoli-like cells. CONCLUSIONS: Our results indicate that isolated 46,XY and 46,XX DSD can be assigned to two separate regulatory regions, XYSR and XXSR, far upstream of SOX9. The 1.9 kb SRY-responsive subfragment from the XYSR might constitute the core of the Sertoli-cell enhancer of human SOX9, representing the so far missing link in the genetic cascade of male sex determination.

Acampomelic form of campomelic dysplasia with SOX9 missense mutation.

Campomelic dysplasia is a skeletal dysplasia characterized by flat face, Pierre Robin sequence, shortening and bowing of long bones and club feet. The authors describe a case of "acampomelic" campomelic dysplasia that differs from classical campomelic dysplasia by the absence of bone bowing. This condition is among the most common skeletal dysplasias but is often misdiagnosed in the absence of overt campomelia.

A case of campomelic dysplasia in whom a new mutation was found in the SOX9 gene.

Campomelic dysplasia (CD, OMIM #114290) is a rare autosomal dominant disease characterized with bending and shortness in the long bones of the lower extremities, typical facial features, hypoplastic scapula, costa defect, narrow thorax and pes equinovarus. Campomelic dysplasia occurs with heterozygous mutations in the SOX9 gene in the 17q24 chromosome. The main findings of our four-day old patient included typical facial features, risomelic extremity shortness, angular bending in the long bones of bilateral lower extremities and pes equinovarus. On direct graphies, costa defect and scapula hypoplasia were noted. We showed a missense mutation (c.473C>T [p.A158V]) in the SOX9 gene which had not been reported before in our patient who had the typical clinical findings of CD. The family of the patient was informed about potential future pathologies of this disease and received genetic counseling.

Scx+/Sox9+ progenitors contribute to the establishment of the junction between cartilage and tendon/ligament.

SRY-box containing gene 9 (Sox9) and scleraxis (Scx) regulate cartilage and tendon formation, respectively. Here we report that murine Scx(+)/Sox9(+) progenitors differentiate into chondrocytes and tenocytes/ligamentocytes to form the junction between cartilage and tendon/ligament. Sox9 lineage tracing in the Scx(+) domain revealed that Scx(+) progenitors can be subdivided into two distinct populations with regard to their Sox9 expression history: Scx(+)/Sox9(+) and Scx(+)/Sox9(-) progenitors. Tenocytes are derived from Scx(+)/Sox9(+) and Scx(+)/Sox9(-) progenitors. The closer the tendon is to the cartilaginous primordium, the more tenocytes arise from Scx(+)/Sox9(+) progenitors. Ligamentocytes as well as the annulus fibrosus cells of the intervertebral discs are descendants of Scx(+)/Sox9(+) progenitors. Conditional inactivation of Sox9 in Scx(+)/Sox9(+) cells causes defective formation in the attachment sites of tendons/ligaments into the cartilage, and in the annulus fibrosus of the intervertebral discs. Thus, the Scx(+)/Sox9(+) progenitor pool is a unique multipotent cell population that gives rise to tenocytes, ligamentocytes and chondrocytes for the establishment of the chondro-tendinous/ligamentous junction.

Tyrosinemia Type III detected via neonatal screening: management and outcome.

Tyrosinemia Type III is caused by the deficiency of 4-hydroxyphenylpyruvate dioxygenase (4-HPPD), an enzyme involved in the catabolic pathway of tyrosine. To our knowledge, only a few patients presenting with this disease have been described in the literature, and the clinical phenotype remains variable and unclear. We report the case of a boy with tyrosinemia Type III detected using neonatal screening, who is homozygous for the splice donor mutation IVS11+1G>A in intron 11 of the HPD gene. At the age of 30 months, the boy's outcome under mild protein restriction was characterized by normal growth and psychomotor development.

Genes promoting and disturbing testis development.

Mammals have an XX/XY sex chromosomal sex determination system in which males represent the heterogametic sex. The Y-linked gene, SRY, determines sex by inducing the undifferentiated, bipotential gonads to differentiate as testes, which produce androgens and promote in this way the development of a male phenotype. Thus, in mammals, sex determination can be equated to testis determination, which involves several important cell processes, including Sertoli cell differentiation, mesonephric cell migration, testis cord formation, testis-specific vascularization, and myoid and Leydig cell differentiation. Many genes are currently known to be involved in testis development. Some of them, including SF1, WT1, GATA4 and FOG2, are necessary for the formation of the bipotential, undifferentiated gonad but also have important roles in testis differentiation. Others can be considered testis-promoting, differentaition and/or maintenance genes: these include SRY, SOX9, FGF9, PTGDS, SOX8, SOX3, NR0B1, PDGFRa, DMRT1, AMH, NGF, NTF3 and NGFR as the most important examples. Finally, there is a smaller group of genes which are involved in ovarian development and which can cause aberrant testis development if mutated, including RSPO1, WNT4, CTNNB1, FST, BMP2 and FOXL2. In this paper, we review our current knowledge on the function, spatio-temporal expression pattern and mutant sexual phenotypes associated with these genes, and discuss the various roles they play in gonad development.

Sox9 and Sox8 are required for basal lamina integrity of testis cords and for suppression of FOXL2 during embryonic testis development in mice.

The sex-determining gene Sry and its target gene Sox9 initiate the early steps of testis development in mammals. Of the related Sox genes Sox8, Sox9, and Sox10, all expressed during Sertoli cell differentiation, only inactivation of Sox9 before the sex determination stage at Embryonic Day 11.5 (E11.5) causes XY sex reversal, while Sox9 inactivation after this stage has no effect on testis cord differentiation. We have previously shown that both Sox9 and Sox8 are essential for maintaining testicular function in post-E14.0 Sertoli cells. To gain insight into the molecular and cellular processes underlying the abnormal development of Sox9 and Sox8 mutant testes, we performed a detailed developmental study of embryonic and neonatal stages. We observe a progressive disruption of the basal lamina surrounding the testis cords that starts at E17.5 and already at E15.5 reduced expression levels of collagen IV, collagen IXa3 and testatin, structural components of the basal lamina, and the extracellular matrix transcriptional regulator Scleraxis. Lineage tracing reveals that mutant Sertoli cells delaminate from testis cords and are present as isolated cells between remaining cords. Also, Sox10 expression is strongly reduced in the absence of Sox9 and/or Sox8. Finally, we document increasing expression of the ovarian marker FOXL2 in mutant cords starting at E15.5, indicating progressive transdifferentiation of mutant Sertoli cells. This study shows that Sox9 and Sox8 maintain integrity of the basal lamina to prevent testis cord disintegration and that both factors actively suppress the ovarian program during early testis development.

L-Sox5 and Sox6 proteins enhance chondrogenic miR-140 microRNA expression by strengthening dimeric Sox9 activity.

Sox9 plays a critical role in early chondrocyte initiation and promotion as well as repression of later maturation. Fellow Sox family members L-Sox5 and Sox6 also function as regulators of cartilage development by boosting Sox9 activation of chondrocyte-specific genes such as Col2a1 and Agc1; however, the regulatory mechanism and other target genes are largely unknown. MicroRNAs are a class of short, non-coding RNAs that act as negative regulators of gene expression by promoting target mRNA degradation and/or repressing translation. Analysis of genetically modified mice identified miR-140 as a cartilage-specific microRNA that could be a critical regulator of cartilage development and homeostasis. Recent findings suggest Sox9 promotes miR-140 expression, although the detailed mechanisms are not fully understood. In this study we demonstrate that the proximal upstream region of pri-miR-140 has chondrogenic promoter activity in vivo. We found an L-Sox5/Sox6/Sox9 (Sox trio) response element and detailed binding site in the promoter region. Furthermore, detailed analysis suggests the DNA binding and/or transactivation ability of Sox9 as a homodimer is boosted by L-Sox5 and Sox6. These findings provide new insight into cartilage-specific gene regulation by the Sox trio.

A large TAT deletion in a tyrosinaemia type II patient.

A girl, born to unrelated Spanish parents, presented at 6 months of age with photophobia, keratitis, palmar hyperkeratosis and high plasma tyrosine levels, indicative of tyrosinaemia type II. Analysis of the tyrosine aminotransferase (TAT) gene revealed a paternally inherited frameshift mutation c.1213delCinsAG at codon 405 causing a premature stop codon, and a maternally inherited deletion of 193kb encompassing the complete TAT gene and three neighbouring genes. This is the first complete TAT deletion in tyrosinaemia type II described so far.

Profiling spermatogenic failure in adult testes bearing Sox9-deficient Sertoli cells identifies genes involved in feminization, inflammation and stress.

BACKGROUND: Sox9 (Sry box containing gene 9) is a DNA-binding transcription factor involved in chondrocyte development and sex determination. The protein's absence in testicular Sertoli nurse cells has been shown to disrupt testicular function in adults but little is known at the genome-wide level about molecular events concomitant with testicular break-down. METHODS: To determine the genome-wide effect on mRNA concentrations triggered by the absence of Sox9 in Sertoli cells we analysed adult testicular tissue from wild-type versus mutant mice with high-density oligonucleotide microarrays and integrated the output of this experiment with regulatory motif predictions and protein-protein network data. RESULTS: We report the genome-wide mRNA signature of adult testes lacking Sox9 in Sertoli cells before and after the onset of late spermatogenic failure as compared to fertile controls. The GeneChip data integrated with evolutionarily conserved Sox9 DNA binding motifs and regulatory network data identified genes involved in feminization, stress response and inflammation. CONCLUSIONS: Our results extend previous observations that genes required for female gonadogenesis are up-regulated in the absence of Sox9 in fetal Sertoli cells to the adult stage. Importantly, we identify gene networks involved in immunological processes and stress response which is reminiscent of a phenomenon occurring in a sub-group of infertile men. This suggests mice lacking Sox9 in their Sertoli cells to be a potentially useful model for adult human testicular failure.

Hydroureternephrosis due to loss of Sox9-regulated smooth muscle cell differentiation of the ureteric mesenchyme.

Congenital ureter anomalies, including hydroureter, affect up to 1% of the newborn children. Despite the prevalence of these developmental abnormalities in young children, the underlying molecular causes are only poorly understood. Here, we show that the high mobility group domain transcription factor Sox9 plays an important role in ureter development in the mouse. Transient Sox9 expression was detected in the undifferentiated ureteric mesenchyme and inactivation of Sox9 in this domain resulted in strong proximal hydroureter formation due to functional obstruction. Loss of Sox9 did not affect condensation, proliferation and apoptosis of the undifferentiated mesenchyme, but perturbed cyto-differentiation into smooth muscle cells (SMCs). Expression of genes encoding extracellular matrix (ECM) components was strongly reduced, suggesting that deficiency in ECM composition and/or signaling may underlie the observed defects. Prolonged expression of Sox9 in the ureteric mesenchyme led to increased deposition of ECM components and SMC dispersal. Furthermore, Sox9 genetically interacts with the T-box transcription factor 18 gene (Tbx18) during ureter development at two levels--as a downstream mediator of Tbx18 function and in a converging pathway. Together, our results argue that obstructive uropathies in campomelic dysplasia patients that are heterozygous for mutations in and around SOX9 arise from a primary requirement of Sox9 in the development of the ureteric mesenchyme.

Mutations of the SRY-responsive enhancer of SOX9 are uncommon in XY gonadal dysgenesis.

During mouse sex determination, SRY upregulates the core testis-specific enhancer of Sox9, TESCO. Mutations in human SRY are found in one third of cases with XY pure gonadal dysgenesis (XY GD; Swyer syndrome), while two thirds remain unexplained. Heterozygous SOX9 mutations can cause XY GD in association with the skeletal malformation syndrome campomelic dysplasia. We hypothesized that human TESCO mutations could cause isolated XY GD. Sixty-six XY GD cases with an intact SRY were analyzed for TESCO point mutations or deletions. No mutations were identified. We conclude that TESCO mutations are not a common cause of XY GD.

Dicer is required for Sertoli cell function and survival.

Dicer is a key enzyme that processes microRNA precursors into their mature form, enabling them to regulate gene expression. Dicer null mutants die before gastrulation. To study Dicer function in testis development, we crossed mice carrying a conditional Dicer allele with an AMH-Cre transgenic line, thereby inactivating Dicer in Sertoli cells around embryonic day 14.0 (E14.0). Dicer null Sertoli cells show normal embryonic development, and at postnatal day 0 (P0), testis tubules are normal in number and histologically undistinguishable from controls. Subsequently, Dicer-mutant testes show a progressively aberrant development, so that at P6, they contain a reduced number of disorganized testis tubules leading to primary sterility. Apoptosis and prophase I assays reveal a massive wave of apoptosis starting at P3, causing progressive loss of Sertoli cells, but also of germ cells, resulting in drastically reduced testis size. Expression of genes that play crucial roles in testis development, structural integrity and spermatogenesis is downregulated at P0, before morphological changes become apparent, indicating that Dicer-mutant testes are already transcriptionally compromised at this stage. Taken together, the results of this study show that Dicer is required for Sertoli cell function and survival and for spermatogenesis in mice.

SOX E genes: SOX9 and SOX8 in mammalian testis development.

The group E SOX proteins consist of SOX8, SOX9 and SOX10. These transcription factors contain, besides a DNA-binding HMG domain and a transactivation domain, a DNA-dependent dimerization domain, unique among SOX proteins. Among these three SOX E proteins, which are all expressed during mammalian testis development, SOX9 stands out in importance. It is SOX9 that becomes activated by SRY in pre-Sertoli cells, executing SRY's role as a testis-determining factor by inducing Sertoli cell and testis cord differentiation. However, Sox9 is dispensable during subsequent embryonic and postnatal testis development, since ablation of Sox9 at embryonic day 14.0, after the sex determination stage, only leads to late-onset sterility at about 5 months. A similar late male sterility phenotype occurs in constitutive Sox8 null mutants. In the combined absence of Sox9 and Sox8, primary male infertility evolves, revealing functional redundancy. Loss of Sox10 has no effect on testis development.