Mutational screening of SF1 and WNT4 in Tunisian women with premature ovarian failure

Background

WNT4 and SF1 genes play an important role in ovarian development. They constitute coherent candidate genes associated with premature ovarian failure (POF) pathogenesis.

Methods

We sequenced the coding region of WNT4 and SF1 in 55 Tunisian women with POF and 100 healthy controls.

Results

We identified a synonymous variation in WNT4 (c.99G>A, p.Ser33Ser) and a substitution (c.G437C) in SF1 gene inducing G146 to Ala (GGG–GCG) missense mutation. WNT4 (c.99G>A, p.Ser33Ser) was not associated with POF pathology. However, a positive association of SF1 Gly146Ala polymorphism was noted. Gly146Ala minor allele frequency was significantly higher (p = 0.029) in POF patients versus controls and Ala allele containing genotypes (p = 0.005) were positively associated with POF pathology. The carriage of 146Ala allele was also associated with a significant reduction in estradiol plasma levels.

Conclusions

SF1 Gly146Ala polymorphism seems to be associated with POF pathology in the Tunisian population likely by reducing estradiol levels.     

Gene expression of WNTs, β-catenin and E-cadherin during the periimplantation period of pregnancy in pigs--involvement of steroid hormones

WNTs (wingless-type MMTV integration site family, member) are morphogenes considered as important factors taking part in uterus developmental processes and implantation. β-catenin is a downstream effector of WNTs action within the cell as well as, through E-cadherin, affecting epithelial organization and function. This study was conducted to investigate WNT4, WNT5A, WNT7A, β-catenin (CTNNB1) and E-cadherin (CDH1) gene expression and protein localization in the endometrium during the periimplantation period. Furthermore, the effect of 17β-estradiol (E₂) and progesterone (P₄) on WNTs, CTNNB1 and CDH1 gene expression in the porcine endometrium in vitro was examined. WNT4 protein was localized in the luminal and glandular epithelium as well as in the basal lamina of the uterine mucosa. WNT5A protein was detected only in the luminal epithelium. WNT7A, β-catenin and E-cadherin protein were identified both in the luminal and glandular epithelial cells, however, WNT7A protein immunoreactivity varied during respective days of estrous cycle and/or pregnancy. Despite unchanged expression of WNT4 mRNA in the endometrium of cyclic and early pregnant pigs, the negative influence of E₂ on WNT4 gene during in vitro experiment was observed. WNT4 and CDH1 gene expression was negatively correlated with blood plasma E₂ and P₄ level in uterine luminal flushings (ULFs) on Day 12 of pregnancy. Expression of WNT5A gene was up-regulated in the endometrium on Day 9 of pregnancy when compared to the respective day of the estrous cycle. A significant decrease of WNT7A gene expression and increase of CDH1 mRNA amount was detected on Day 12 of pregnancy. Overall, the results show the spatial localization of WNT4, WNT5A, WNT7A, β-catenin and E-cadherin proteins in porcine endometrium during periimplantation period of pregnancy and indicate significant changes of WNT5A, WNT7A and CDH1 gene expression before implantation in the pig.     

Involvement of and Interaction between WNT10A and EDA Mutations in Tooth Agenesis Cases in the Chinese Population

Background

Dental agenesis is the most common, often heritable, developmental anomaly in humans. Although WNT10A gene mutations are known to cause rare syndromes associated with tooth agenesis, including onycho-odontodermal dysplasia (OODD), Schöpf-Schulz-Passarge syndrome (SSPS), hypohidrotic ectodermal dysplasia (HED), and more than half of the cases of isolated oligodontia recently, the genotype-phenotype correlations and the mode of inheritance of WNT10A mutations remain unclear. The phenotypic expression with WNT10A mutations shows a high degree of variability, suggesting that other genes might function with WNT10A in regulating ectodermal organ development. Moreover, the involvement of mutations in other genes, such as EDA, which is also associated with HED and isolated tooth agenesis, is not clear. Therefore, we hypothesized that EDA mutations interact with WNT10A mutations to play a role in tooth agenesis. Additionally, EDA, EDAR, and EDARADD encode signaling molecules in the Eda/Edar/NF-κB signaling pathways, we also checked EDAR and EDARADD in this study.

Methods

WNT10A, EDA, EDAR and EDARADD were sequenced in 88 patients with isolated oligodontia and 26 patients with syndromic tooth agenesis. The structure of two mutated WNT10A and two mutated EDA proteins was analyzed.

Results

Digenic mutations of both WNT10A and EDA were identified in 2 of 88 (2.27%) isolated oligodontia cases and 4 of 26 (15.38%) syndromic tooth agenesis cases. No mutation in EDAR or EDARADD gene was found.

Conclusions

WNT10A and EDA digenic mutations could result in oligodontia and syndromic tooth agenesis in the Chinese population. Moreover, our results will greatly expand the genotypic spectrum of tooth agenesis.     

The WNT7A G204S mutation is associated with both Al-Awadi–Raas Rothschild syndrome and Fuhrmann syndrome phenotypes

Two syndromes are known to be associated with WNT7A mutations: Al-Awadi–Raas-Rothschild syndrome (AARRS) and Fuhrmann syndrome. Woods et al. (2006) showed that there is complete and partial loss of WNT7A function in these two syndromes respectively. Therefore, both syndromes have similar clinical features but the phenotype in Fuhrmann syndrome is less severe. The G204S mutation was previously reported to result in AARRS phenotype in three Saudi families. In the current communication, we report on a different unrelated Saudi patient with the same mutation but the patient had Fuhrmann syndrome phenotype. We believe this case is important because it questions the presence of a phenotype–genotype correlation in WNT7A mutations and because it demonstrates that the G204S mutation may be associated with both AARRS and Fuhrmann phenotypes.     

Inhibition of Cancer Cell Migration and Invasion through Suppressing the Wnt1-mediating Signal Pathway by G-quadruplex Structure Stabilizers

WNT1 encodes a multifunctional signaling glycoprotein that is highly expressed in several malignant tumors. Patients with Wnt1-positive cancer are usually related to advanced metastasis. Here, we found that a stretch of G-rich sequences located at the WNT1 promoter region is capable of forming G-quadruplex structures. The addition of G-quadruplex structure stabilizers, BMVC and BMVC4, raises the melting temperature of the oligonucleotide formed by the WNT1 promoter G-rich sequences. Significantly, the expression of WNT1 was repressed by BMVC or BMVC4 in a G-quadruplex-dependent manner, suggesting that they can be used to modulate WNT1 expression. The role of G-quadruplex stabilizers on Wnt1-mediated cancer migration and invasion was further analyzed. The protein levels of β-catenin, a mediator of the Wnt-mediated signaling pathway, and the downstream targets MMP7 and survivin were down-regulated upon BMVC or BMVC4 treatments. Moreover, the migration and invasion activities of cancer cells were inhibited by BMVC and BMVC4, and the inhibitory effects can be reversed by WNT1-overexpression. Thus the Wnt1 expression and its downstream signaling pathways can be regulated through the G-quadruplex sequences located at its promoter region. These findings provide a novel approach for future drug development to inhibit migration and invasion of cancer cells.     

Dullard/Ctdnep1 Modulates WNT Signalling Activity for the Formation of Primordial Germ Cells in the Mouse Embryo

Dullard/Ctdnep1 is a member of the serine/threonine phosphatase family of the C-terminal domain of eukaryotic RNA polymerase II. Embryos lacking Dullard activity fail to form primordial germ cells (PGCs). In the mouse, the formation of PGCs is influenced by BMP4 and WNT3 activity. Although Dullard is reputed to negatively regulate BMP receptor function, in this study we found mutations in Dullard had no detectable effect on BMP4 and p-Smad activity. Furthermore Dullard mutations did not influence the dosage-dependent inductive effect of Bmp4 in PGC formation. However, Dullard may function as a positive regulator of WNT signalling. Combined loss of one copy each of Dullard and Wnt3 had a synergistic effect on the reduction of PGC numbers in the compound heterozygous embryo. In addition, loss of Dullard function was accompanied by down-regulation of WNT/β-catenin signalling activity and a reduction in the level of Dishevelled 2 (Dvl2). Therefore, Dullard may play a role in the fine-tuning of WNT signalling activity by modulating the expression of ligands/antagonists and the availability of Dvl2 protein during specification of the germ cell lineage.     

Novel mutation G324C in <Emphasis Type="Italic">WNT1</Emphasis> mapped in a large Pakistani family with severe recessively inherited <Emphasis Type="Italic">Osteogenesis Imperfecta</Emphasis>

Introduction

Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous disease with skeletal fragility and variable extra-skeletal manifestations. To date several point mutations in 18 different genes causing different types of OI have been identified. Mutations in WNT1 compromise activity of the osteoblasts leading to disturbed bone mass accrual, fragility fractures and progressive skeletal abnormalities. The present study was conducted to determine the underlying genetic cause of an autosomal recessive skeletal dysplasia in a large consanguineous family from Chinute, Pakistan.

Materials and methods

Blood was collected from 24 individuals of affected family along with clinical data. Homozygosity mapping was performed to confirm consanguinity. SNPs were identified, followed by whole exome and Sanger sequencing. In silico characterization of WNT1 mutation was performed using multiple platforms.

Results

Nine affected family members exhibited severe bone deformities, recurrent fractures, short stature and low bone mineral density. SNP array data revealed homozygous segments >?1 Mb in length accounting for 2.1–12.7% of the genome in affected individuals and their siblings and a single 6,344,821 bp homozygous region in all affected individuals on chromosome 12q12-q13. This region includes two potential OI candidate genes WNT1 and VDR. We did whole-exome sequencing for both genes in two patients and identified a novel damaging missense mutation in exon 4 of WNT1: c.1168G?>?T (NM_005430) resulting in p.G324C. Sanger sequencing confirmed segregation of mutation with the disease in family.

Conclusion

We report a novel mutation responsible for OI and our investigation expands the spectrum of disease-causing WNT1 mutations and the resulting OI phenotypes.

     

WNT10A Mutations Are a Frequent Cause of a Broad Spectrum of Ectodermal Dysplasias with Sex-Biased Manifestation Pattern in Heterozygotes

Odonto-onycho-dermal dysplasia (OODD), a rare autosomal-recessive inherited form of ectodermal dysplasia including severe oligodontia, nail dystrophy, palmoplantar hyperkeratosis, and hyperhidrosis, was recently shown to be caused by a homozygous nonsense WNT10A mutation in three consanguineous Lebanese families. Here, we report on 12 patients, from 11 unrelated families, with ectodermal dysplasia caused by five previously undescribed WNT10A mutations. In this study, we show that (1) WNT10A mutations cause not only OODD but also other forms of ectodermal dysplasia, reaching from apparently monosymptomatic severe oligodontia to Schöpf-Schulz-Passarge syndrome, which is so far considered a unique entity by the findings of numerous cysts along eyelid margins and the increased risk of benign and malignant skin tumors; (2) WNT10A mutations are a frequent cause of ectodermal dysplasia and were found in about 9% of an unselected patient cohort; (3) about half of the heterozygotes (53.8%) show a phenotype manifestation, including mainly tooth and nail anomalies, which was not reported before in OODD; and (4) heterozygotes show a sex-biased manifestation pattern, with a significantly higher proportion of tooth anomalies in males than in females, which may implicate gender-specific differences of WNT10A expression.     

Chronic Academic Stress Increases a Group of microRNAs in Peripheral Blood

MicroRNAs (miRNAs) play key roles in regulation of cellular processes in response to changes in environment. In this study, we examined alterations in miRNA profiles in peripheral blood from 25 male medical students two months and two days before the National Examination for Medical Practitioners. Blood obtained one month after the examination were used as baseline controls. Levels of seven miRNAs (miR-16, -20b, -26b, -29a, -126, -144 and -144*) were significantly elevated during the pre-examination period in association with significant down-regulation of their target mRNAs (WNT4, CCM2, MAK, and FGFR1 mRNAs) two days before the examination. State anxiety assessed two months before the examination was positively and negatively correlated with miR-16 and its target WNT4 mRNA levels, respectively. Fold changes in miR-16 levels from two days before to one month after the examination were inversely correlated with those in WNT4 mRNA levels over the same time points. We also confirmed the interaction between miR-16 and WNT4 3′UTR in HEK293T cells overexpressing FLAG-tagged WNT4 3′UTR and miR-16. Thus, a distinct group of miRNAs in periheral blood may participate in the integrated response to chronic academic stress in healthy young men.     

Mutation analysis of the WNT4 gene in Han Chinese women with premature ovarian failure

Background

The WNT4 gene plays an important role in female sex determination and differentiation. It also contributes to maintaining of the ovaries and the survival of follicles.

Methods

We sequenced the coding region and splice sites of WNT4 in 145 Han Chinese women with premature ovarian failure (POF) and 200 healthy controls.

Results

Only one novel variation, in Exon 2 (195C > T), was detected among the women with POF. However, this synonymous variation did not result in a change in amino acid sequence (65 Asp > Asp). No further variants were found in any of the samples.

Conclusion

Although we cannot provide any evidence that it is a possible disease-causing gene, this study is the first attempt to investigate the possible role of WNT4 in Han Chinese women with POF.     

WNT10A variants are associated with non-syndromic tooth agenesis in the general population

Tooth agenesis is the most common developmental dental anomaly. Absence of one or two permanent teeth is found in the majority of affected subjects. Very few patients suffer severe tooth agenesis. Recent studies revealed that WNT10A gene mutations caused syndromic and isolated severe tooth agenesis. In this study, to determine the contribution of WNT10A variants in different severities of tooth agenesis, we investigated the association between WNT10A variants and non-syndromic tooth agenesis in a Chinese population consisting of 505 tooth agenesis patients and 451 normal controls. Twenty-three novel non-synonymous variants were identified. WNT10A variants were detected in 15.8 % (75/474) of patients with 1–3 missing teeth and 51.6 % (16/31) of patients with 4 or more missing teeth. As compared with a frequency of 3.1 % in individuals with full dentition, variant allele frequencies were significantly elevated in both groups with tooth agenesis (p values of 1.00 × 10^?6 and 3.89 × 10^?23, respectively). Our findings showed that WNT10A variants were associated with non-syndromic tooth agenesis from mild to severe tooth agenesis, and the more severe tooth agenesis, the stronger association. Biallelic genotypes of WNT10A variants may have a pathogenic effect on tooth development. Presence of a single variant allele would be predisposing for causation with low penetrance. Together with WNT10A variant, there should be other genetic or environmental factors leading to biallelic variant-related variable clinical manifestations and single allele variant-related low penetrance. The frequent missing tooth positions in the WNT10A-related cases were consistent with that in the general population, suggesting WNT10A plays a critically important role in the etiology of general tooth agenesis.     

Regulation of the Follistatin Gene by RSPO-LGR4 Signaling via Activation of the WNT/β-Catenin Pathway in Skeletal Myogenesis

WNT signaling plays multiple roles in skeletal myogenesis during gestation and postnatal stages. The R-spondin (RSPO) family of secreted proteins and their cognate receptors, members of leucine-rich repeat-containing G protein-coupled receptor (LGR) family, have emerged as new regulatory components of the WNT signaling pathway. We previously showed that RSPO2 promoted myogenic differentiation via activation of WNT/β-catenin signaling in mouse myoblast C2C12 cells in vitro. However, the molecular mechanism by which RSPO2 regulates myogenic differentiation is unknown. Herein, we show that depletion of the LGR4 receptor severely disrupts myogenic differentiation and significantly diminishes the response to RSPO2 in C2C12 cells, showing a requirement of LGR4 in RSPO signaling during myogenic differentiation. We identify the transforming growth factor β (TGF-β) antagonist follistatin (Fst) as a key mediator of RSPO-LGR4 signaling in myogenic differentiation. We further demonstrate that Fst is a direct target of the WNT/β-catenin pathway. Activation and inactivation of β-catenin induced and inhibited Fst expression, respectively, in both C2C12 cells and mouse embryos. Specific TCF/LEF1 binding sites within the promoter and intron 1 region of the Fst gene were required for RSPO2 and WNT/β-catenin-induced Fst expression. This study uncovers a molecular cross talk between WNT/β-catenin and TGF-β signaling pivotal in myogenic differentiation.     

WNT5A selectively inhibits mouse ventral prostate development

The establishment of prostatic budding patterns occurs early in prostate development but mechanisms responsible for this event are poorly understood. We investigated the role of WNT5A in patterning prostatic buds as they emerge from the fetal mouse urogenital sinus (UGS). Wnt5a mRNA was expressed in UGS mesenchyme during budding and was focally up-regulated as buds emerged from the anterior, dorsolateral, and ventral UGS regions. We observed abnormal UGS morphology and prostatic bud patterns in Wnt5a null male fetuses, demonstrated that prostatic bud number was decreased by recombinant mouse WNT5A protein during wild type UGS morphogenesis in vitro, and showed that ventral prostate development was selectively impaired when these WNT5A-treated UGSs were grafted under under kidney capsules of immunodeficient mice and grown for 28 d. Moreover, a WNT5A inhibitory antibody, added to UGS organ culture media, rescued prostatic budding from inhibition by a ventral prostatic bud inhibitor, 2,3,8,7-tetrachlorodibenzo-p-dioxin, and restored ventral prostate morphogenesis when these tissues were grafted under immunodeficient mouse kidney capsules and grown for 28 d. These results suggest that WNT5A participates in prostatic bud patterning by restricting mouse ventral prostate development.     

Further evidence of the involvement of the Wnt signaling pathway in Dupuytren’s disease

Genetic background plays an important role in the development of Dupuytren’s disease. A genome-wide association study (GWAS) showed that nine loci are associated with the disease, six of which contain genes that are involved in Wnt signaling (WNT2, WNT4, WNT7B, RSPO2, SFRP4, SULF1). To obtain insight in the role of these genes, we performed expression studies on affected and unaffected patient’s tissues. Surgically obtained nodules and cords from eight Dupuytren’s patients were compared to patient-matched control tissue (unaffected transverse palmar fascia). The Wnt-related genes found in the GWAS, the classical Wnt-downstream protein β-catenin, as well as (myo)fibroblast markers were analyzed using real-time qPCR and immunohistochemical stainings for mRNA levels and protein levels, respectively. The collagen-coding genes COL1A1 and COL3A1 were highly upregulated on mRNA level, both in cords and nodules. Three Wnt-related genes were found to be differently regulated compared to control tissue: WNT2 was downregulated in nodules, WNT7B was upregulated in nodules, and SFRP4 was upregulated in nodules and cords. Immunohistochemistry revealed significantly less staining of Wnt2 in cords, but significantly more staining for Wnt7b in nodules. There was significantly more staining of α-SMA in nodules and cord and β-catenin in nodules than in control tissue. We found differences in expression, both at mRNA and protein level, in several Wnt-related genes found earlier to be associated with Dupuytren’s disease. Of these, Wnt7b was upregulated and found in close association with both α-SMA and β-catenin expressing cells, making it a candidate pro-fibrotic mediator in Dupuytren’s disease.