Cryptophthalmos: associated syndromes and genetic disorders.

PURPOSE: Cryptophthalmos is a rare congenital condition caused by anomalous eyelid development where the eyelid folds do not develop or fail to separate. Cryptophthalmos can be unilateral or bilateral and can occur in isolation or as part of an underlying syndrome. We aim to identify genetic syndromes associated with cryptophthalmos to facilitate genetic diagnosis. METHODS: We performed a retrospective medical record review of all patients diagnosed with cryptophthalmos followed at a single center between 2000 and 2020. The analysis included medical history, clinical examination findings, and genetic testing results. RESULTS: Thirteen patients were included, 10 (77%) males, mean age of 2.4 years. Eight (61%) had bilateral cryptophthalmos, and 4 (31%) had complete cryptophthalmos. Associated ocular abnormalities included corneal opacities (13/13, 100%), upper eyelid colobomas (12/13, 92%), and microphthalmia/clinical anophthalmia (3/13, 23%). All cases of complete cryptophthalmos had bilateral disease. An underlying clinical or molecular diagnosis was identified in 10/13 (77%) cases, including Fraser syndrome (n = 5), amniotic band syndrome (n = 1), FREM1-related disease (n = 1), Goldenhar versus Schimmelpenning syndrome (n = 1), MOTA syndrome (n = 1), and CELSR2-related disease (n = 1). CONCLUSION: This is the first report of a possible association between cryptophthalmos and biallelic CELSR2 variants. Children with cryptophthalmos, especially those with extra-ocular involvement, should be referred for comprehensive genetic evaluation.

FREM2-related Fraser syndrome with popliteal pterygium and structural central nervous system anomalies.

Fraser syndrome (FS) is a rare multiple malformation disorder characterized by cryptophthalmos, characteristic craniofacial dysmorphism, cutaneous syndactyly, malformations of the respiratory and urinary tract, and anogenital anomalies. Although the characteristic presentation of FS can be detected prenatally, oligohydramnios often challenges the clinical diagnosis. Here we report on the atypical prenatal and postmortem findings of a fetus with FS caused by a novel homozygous frameshift variant in FREM2. Our study highlights the variable manifestations of the FS and expands the clinical spectrum to include popliteal pterygium and structural central nervous system anomalies.

Identification and Comprehensive Analysis of FREM2 Mutation as a Potential Prognostic Biomarker in Colorectal Cancer.

Gene mutations play an important role in tumor progression. This study aimed to identify genes that were mutated in colorectal cancer (CRC) and to explore their biological effects and prognostic value in CRC patients. We performed somatic mutation analysis using data sets from The Cancer Genome Atlas and International Cancer Genome Consortium, and identified that FREM2 had the highest mutation frequency in patients with colon adenocarcinoma (COAD). COAD patients were divided into FREM2-mutated type (n = 36) and FREM2-wild type (n = 278), and a Kaplan-Meier survival curve was generated to perform prognostic analysis. A FREM2-mutation prognosis model was constructed using random forest method, and the performance of the model was evaluated using receiver operating characteristic curve. Next, the random forest method and Cox regression analysis were used to construct a prognostic model based on the gene expression data of 36 FREM2-mutant COAD patients. The model showed a high prediction accuracy (83.9%), and 13 prognostic model characteristic genes related to overall survival were identified. Then, the results of tumor mutation burden (TMB) and microsatellite instability (MSI) analyses revealed significant differences in TMB and MSI among the risk scores of different prognostic models. Differentially expressed genes were identified and analyzed for functional enrichment and immune infiltration. Finally, 30 samples of CRC patients were collected for immunohistochemical staining to analyze the FREM2 expression levels, which showed that FREM2 was highly expressed in tumor tissues. In conclusion, CRC patients had a high level of FREM2 mutations associated with a worse prognosis, which indicated that FREM2 mutations may be potential prognostic markers in CRC.

Excluding embryos with two novel mutations in FREM2 gene by the next-generation sequencing-based single nucleotide polymorphism haplotyping.

Fraser syndrome is a rare autosomal recessive malformation disorder. It is characterized by cryptophthalmos, syndactyly, urinary tract abnormalities and ambiguous genitalia. This condition is due to homozygous or heterozygous mutations in the FRAS1, FREM1, FREM2, and GRIP1 genes. In the present study, we recruited a Chinese family with Fraser syndrome. Two novel mutations c.7542_7543insG and c.2689C>T in the FREM2 gene were detected in this Fraser syndrome family by PCR-based sequencing. The next-generation sequencing-based single nucleotide polymorphism haplotyping method was applied to exclude these two mutations in 9 blastocysts obtained from the patient. After obtaining consent and informing the risk, the patient received in vitro fertilization and embryo transfer treatment with an embryo carrying a heterozygous mutation. Finally, she delivered a healthy baby without any complications on March 17, 2019. In conclusion, we first reported two novel mutations in the FREM2 gene associated with the risk of Fraser syndrome. Moreover, we described a next-generation sequencing-based single nucleotide polymorphism haplotyping method to select the 'right' embryos from patients with Fraser syndrome for in vitro fertilization and embryo transfer treatment.

Algorithmically Deduced FREM2 Molecular Pathway Is a Potent Grade and Survival Biomarker of Human Gliomas.

Gliomas are the most common malignant brain tumors with high mortality rates. Recently we showed that the FREM2 gene has a role in glioblastoma progression. Here we reconstructed the FREM2 molecular pathway using the human interactome model. We assessed the biomarker capacity of FREM2 expression and its pathway as the overall survival (OS) and progression-free survival (PFS) biomarkers. To this end, we used three literature and one experimental RNA sequencing datasets collectively covering 566 glioblastomas (GBM) and 1097 low-grade gliomas (LGG). The activation level of deduced FREM2 pathway showed strong biomarker characteristics and significantly outperformed the FREM2 expression level itself. For all relevant datasets, it could robustly discriminate GBM and LGG (p < 1.63 × 10-13, AUC > 0.74). High FREM2 pathway activation level was associated with poor OS in LGG (p < 0.001), and low PFS in LGG (p < 0.001) and GBM (p < 0.05). FREM2 pathway activation level was poor prognosis biomarker for OS (p < 0.05) and PFS (p < 0.05) in LGG with IDH mutation, for PFS in LGG with wild type IDH (p < 0.001) and mutant IDH with 1p/19q codeletion(p < 0.05), in GBM with unmethylated MGMT (p < 0.05), and in GBM with wild type IDH (p < 0.05). Thus, we conclude that the activation level of the FREM2 pathway is a potent new-generation diagnostic and prognostic biomarker for multiple molecular subtypes of GBM and LGG.

Human disease-associated extracellular matrix orthologs ECM3 and QBRICK regulate primary mesenchymal cell migration in sea urchin embryos.

Sea urchin embryos have been one of model organisms to investigate cellular behaviors because of their simple cell composition and transparent body. They also give us an opportunity to investigate molecular functions of human proteins of interest that are conserved in sea urchin. Here we report that human disease-associated extracellular matrix orthologues ECM3 and QBRICK are necessary for mesenchymal cell migration during sea urchin embryogenesis. Immunofluorescence has visualized the colocalization of QBRICK and ECM3 on both apical and basal surface of ectoderm. On the basal surface, QBRICK and ECM3 constitute together a mesh-like fibrillar structure along the blastocoel wall. When the expression of ECM3 was knocked down by antisense-morpholino oligonucleotides, the ECM3-QBRICK fibrillar structure completely disappeared. When QBRICK was knocked down, the ECM3 was still present, but the basally localized fibers became fragmented. The ingression and migration of primary mesenchymal cells were not critically affected, but their migration at later stages was severely affected in both knock-down embryos. As a consequence of impaired primary mesenchymal cell migration, improper spicule formation was observed. These results indicate that ECM3 and QBRICK are components of extracellular matrix, which play important role in primary mesenchymal cell migration, and that sea urchin is a useful experimental animal model to investigate human disease-associated extracellular matrix proteins.

Novel loss of function variants in FRAS1 AND FREM2 underlie renal agenesis in consanguineous families.

INTRODUCTION: Congenital anomalies of the kidney and urinary tract (CAKUT) are a group of abnormalities that affect structure of the kidneys or other structures of the urinary tract. The majority of CAKUT are asymptomatic and are diagnosed prenatally by ultrasound scanning or found incidentally in postnatal life. CAKUT varies in severity and may lead to life-threatening kidney failure and end-stage kidney disease. Renal agenesis, a severe form of CAKUT, is a congenital absence of one or both kidneys. Bilateral renal agenesis belongs to a group of prenatally lethal renal diseases and is often detected on fetal ultrasound scanning during the investigation of oligohydramnios. Approximately 40% of fetuses with bilateral renal agenesis are stillborn or die a few hours postnatally. Mutations in many renal development genes have been shown to be associated with renal agenesis. METHODS: Six consanguineous Saudi Arabian families were recruited to study the molecular genetic causes of recurrent miscarriages and lost fetuses due to oligohydramnios, renal agenesis and other congenital anomalies. Whole exome sequencing was employed to underlying detect genetic defects. RESULTS: Novel loss of function variants were detected in FRAS1 and FREM2. In FRAS1, a homozygous splice site variant c.9780+2T>C was found in an affected fetus, segregating form each parent. In addition, in three other families both parents were heterozygous for a frameshift variant (c.8981dupT; p.His2995Profs*3) and splice site variants (c.5217+1G>C and c.8098+2T>A), respectively. In FREM2, a homozygous nonsense variant (c.2303C>G; p.Ser768*) was found in an affected fetus, segregating from both parents. In another family, both parents carried a FREM2 heterozygous frameshift variant (c.3969delC; p.Asn1323Lysfs*5). CONCLUSION: We describe consanguineous families with clinical features of antenatal oligohydramnios and bilateral renal agenesis, in whom we have identified novel pathogenic variants in FRAS1 and FREM2. These finding highlights the association between mutations in FRAS1 and FREM2 and antenatal/perinatal death.

The Metabolic Reprogramming of Frem2 Mutant Mice Embryos in Cryptophthalmos Development.

BACKGROUND: Cryptophthalmos is characterized by congenital ocular dysplasia with eyelid malformation. The pathogenicity of mutations in genes encoding components of the FRAS1/FREM protein complex is well established, but the underlying pathomechanisms of this disease are still unclear. In the previous study, we generated mice carrying Frem2 R725X/R2156W compound heterozygous mutations using CRISPR/Cas9 and showed that these mice recapitulated the human cryptophthalmos phenotype. METHODS: In this study, we tracked changes in the metabolic profile of embryos and expression of metabolism-related genes in Frem2 mutant mice on E13.5 compared with wild-type mice. RNA sequencing (RNA-seq) was utilized to decipher the differentiated expression of genes associated with metabolism. Untargeted metabolomics and targeted metabolomics analyses were performed to detect and verify the shifts in the composition of the embryonic metabolome. RESULTS: Differentially expressed genes participating in amino acid metabolism and energy metabolism were observed by RNA-seq. Transcriptomic analysis suggests that 821 (39.89%) up-regulated genes and 320 (32.99%) down-regulated genes were involved in the metabolic process in the enriched GO terms. A total of 92 significantly different metabolites were identified including creatine, guanosine 5'-monophosphate, cytosine, cytidine 5'-monophosphate, adenine, and L-serine. Interestingly, major shifts related to ATP binding cassette transporters (ABC transporters) and the biosynthesis of amino acids in the composition of the embryonic metabolome were observed by KEGG metabolic analysis, indicating that these pathways could also be involved in the pathogenesis of cryptophthalmos. CONCLUSION: We demonstrate that Frem2 mutant fetal mice have increased susceptibility to the disruption of eye morphogenesis in association with distinct transcriptomic and metabolomic signatures. Our findings suggest that the metabolomic signature established before birth may play a role in mediating cryptophthalmos in Frem2 mutant mice, which may have important implications for the pathogenesis of cryptophthalmos.

High FREM2 Gene and Protein Expression Are Associated with Favorable Prognosis of IDH-WT Glioblastomas.

World Health Organization grade IV diffuse gliomas, known as glioblastomas, are the most common malignant brain tumors, and they show poor prognosis. Multimodal treatment of surgery followed by radiation and chemotherapy is not sufficient to increase patient survival, which is 12 to 18 months after diagnosis. Despite extensive research, patient life expectancy has not significantly improved over the last decade. Previously, we identified FREM2 and SPRY1 as genes with differential expression in glioblastoma cell lines compared to nonmalignant astrocytes. In addition, the FREM2 and SPRY1 proteins show specific localization on the surface of glioblastoma cells. In this study, we explored the roles of the FREM2 and SPRY1 genes and their proteins in glioblastoma pathology using human tissue samples. We used proteomic, transcriptomic, and bioinformatics approaches to detect changes at different molecular levels. We demonstrate increased FREM2 protein expression levels in glioblastomas compared to reference samples. At the transcriptomic level, both FREM2 and SPRY1 show increased expression in tissue samples of different glioma grades compared to nonmalignant brain tissue. To broaden our experimental findings, we analyzed The Cancer Genome Atlas glioblastoma patient datasets. We discovered higher FREM2 and SPRY1 gene expression levels in glioblastomas compared to lower grade gliomas and reference samples. In addition, we observed that low FREM2 expression was associated with progression of IDH-mutant low-grade glioma patients. Multivariate analysis showed positive association between FREM2 and favorable prognosis of IDH-wild type glioblastoma. We conclude that FREM2 has an important role in malignant progression of glioblastoma, and we suggest deeper analysis to determine its involvement in glioblastoma pathology.

Loss-of-function mutations in FREM2 disrupt eye morphogenesis.

Cryptophthalmos is a rare congenital disorder characterized by ocular dysplasia with eyelid malformation. Complete cryptophthalmos is characterized by the presence of continuous skin from the forehead over the eyes and onto the cheek, along with complete fusion of the eyelids. In the present study, we characterized the clinical manifestations of three patients with isolated bilateral cryptophthalmos. These patients shared the same c.6499C > T missense mutation in the FRAS1-related extracellular matrix protein 2 (FREM2) gene, while each individual presented an additional nonsense mutation in the same gene (Patient #1, c.2206C > T; Patient #2, c.5309G > A; and Patient #3, c.4063C > T). Then, we used CRISPR/Cas9 to generate mice carrying Frem2R725X/R2156W compound heterozygous mutations, and showed that these mice recapitulated the human isolated cryptophthalmos phenotype. We detected FREM2 expression in the outer plexiform layer of the retina for the first time in the cryptophthalmic eyes, and the levels were comparable to the wild-type mice. Moreover, a set of different expressed genes that may contribute secondarily to the phenotypes were identified by performing RNA sequencing (RNA-seq) of the fetal Frem2 mutant mice. Our findings extend the spectrum of FREM2 mutations, and provide insights into opportunities for the prenatal diagnosis of isolated cryptophthalmos. Furthermore, our work highlights the importance of the FREM2 protein during the development of eyelids and the anterior segment of the eyeballs, establishes a suitable animal model for studying epithelial reopening during eyelid development and serves as a valuable reference for further mechanistic studies of the pathogenesis of isolated cryptophthalmos.

Meta-Analysis and Experimental Validation Identified FREM2 and SPRY1 as New Glioblastoma Marker Candidates.

Glioblastoma (GB) is the most aggressive brain malignancy. Although some potential glioblastoma biomarkers have already been identified, there is a lack of cell membrane-bound biomarkers capable of distinguishing brain tissue from glioblastoma and/or glioblastoma stem cells (GSC), which are responsible for the rapid post-operative tumor reoccurrence. In order to find new GB/GSC marker candidates that would be cell surface proteins (CSP), we have performed meta-analysis of genome-scale mRNA expression data from three data repositories (GEO, ArrayExpress and GLIOMASdb). The search yielded ten appropriate datasets, and three (GSE4290/GDS1962, GSE23806/GDS3885, and GLIOMASdb) were used for selection of new GB/GSC marker candidates, while the other seven (GSE4412/GDS1975, GSE4412/GDS1976, E-GEOD-52009, E-GEOD-68848, E-GEOD-16011, E-GEOD-4536, and E-GEOD-74571) were used for bioinformatic validation. The selection identified four new CSP-encoding candidate genes—CD276, FREM2, SPRY1, and SLC47A1—and the bioinformatic validation confirmed these findings. A review of the literature revealed that CD276 is not a novel candidate, while SLC47A1 had lower validation test scores than the other new candidates and was therefore not considered for experimental validation. This validation revealed that the expression of FREM2—but not SPRY1—is higher in glioblastoma cell lines when compared to non-malignant astrocytes. In addition, FREM2 gene and protein expression levels are higher in GB stem-like cell lines than in conventional glioblastoma cell lines. FREM2 is thus proposed as a novel GB biomarker and a putative biomarker of glioblastoma stem cells. Both FREM2 and SPRY1 are expressed on the surface of the GB cells, while SPRY1 alone was found overexpressed in the cytosol of non-malignant astrocytes.

Mesenchymal expression of the FRAS1/FREM2 gene unit is decreased in the developing fetal diaphragm of nitrofen-induced congenital diaphragmatic hernia.

PURPOSE: Developmental mutations that inhibit normal formation of extracellular matrix (ECM) in fetal diaphragms have been identified in congenital diaphragmatic hernia (CDH). FRAS1 and FRAS1-related extracellular matrix 2 (FREM2), which encode important ECM proteins, are secreted by mesenchymal cells during diaphragmatic development. The FRAS1/FREM2 gene unit has been shown to form a ternary complex with FREM1, which plays a crucial role during formation of human and rodent diaphragms. Furthermore, it has been demonstrated that the diaphragmatic expression of FREM1 is decreased in the nitrofen-induced CDH model. We hypothesized that FRAS1 and FREM2 expression is decreased in the developing diaphragms of fetal rats with nitrofen-induced CDH. METHODS: Pregnant rats were exposed to either nitrofen or vehicle on gestational day 9 (D9), and fetuses were harvested on D13, D15 and D18. Microdissected diaphragms were divided into nitrofen-exposed/CDH and control samples (n = 12 per time-point and experimental group, respectively). Diaphragmatic gene expression levels of FRAS1 and FREM2 were analyzed by qRT-PCR. Immunofluorescence double staining for FRAS1 and FREM2 was combined with the mesenchymal marker GATA4 in order to evaluate protein expression and localization in pleuroperitoneal folds (PPFs) and fetal diaphragmatic tissue. RESULTS: Relative mRNA expression of FRAS1 and FREM2 were significantly reduced in PPFs of nitrofen-exposed fetuses on D13 (1.76 ± 0.86 vs. 3.09 ± 1.15; p < 0.05 and 0.47 ± 0.26 vs. 0.82 ± 0.36; p < 0.05), developing diaphragms of nitrofen-exposed fetuses on D15 (1.45 ± 0.80 vs. 2.63 ± 0.84; p < 0.05 and 0.41 ± 0.16 vs. 1.02 ± 0.49; p < 0.05) and fully muscularized diaphragms of CDH fetuses on D18 (1.35 ± 0.75 vs. 2.32 ± 0.92; p < 0.05 and 0.37 ± 0.24 vs. 0.70 ± 0.32; p < 0.05) compared to controls. Confocal laser scanning microscopy revealed markedly diminished FRAS1 and FREM2 immunofluorescence in diaphragmatic mesenchyme, which was associated with reduced proliferation of mesenchymal cells in nitrofen-exposed PPFs and fetal CDH diaphragms on D13, D15 and D18 compared to controls. CONCLUSION: Decreased mesenchymal expression of FRAS1 and FREM2 in the nitrofen-induced CDH model may cause failure of the FRAS1/FREM2 gene unit to activate FREM1 signaling, disturbing the formation of diaphragmatic ECM and thus contributing to the development of diaphragmatic defects in CDH.

Mild recessive mutations in six Fraser syndrome-related genes cause isolated congenital anomalies of the kidney and urinary tract.

Congenital anomalies of the kidney and urinary tract (CAKUT) account for approximately 40% of children with ESRD in the United States. Hitherto, mutations in 23 genes have been described as causing autosomal dominant isolated CAKUT in humans. However, >90% of cases of isolated CAKUT still remain without a molecular diagnosis. Here, we hypothesized that genes mutated in recessive mouse models with the specific CAKUT phenotype of unilateral renal agenesis may also be mutated in humans with isolated CAKUT. We applied next-generation sequencing technology for targeted exon sequencing of 12 recessive murine candidate genes in 574 individuals with isolated CAKUT from 590 families. In 15 of 590 families, we identified recessive mutations in the genes FRAS1, FREM2, GRIP1, FREM1, ITGA8, and GREM1, all of which function in the interaction of the ureteric bud and the metanephric mesenchyme. We show that isolated CAKUT may be caused partially by mutations in recessive genes. Our results also indicate that biallelic missense mutations in the Fraser/MOTA/BNAR spectrum genes cause isolated CAKUT, whereas truncating mutations are found in the multiorgan form of Fraser syndrome. The newly identified recessive biallelic mutations in these six genes represent the molecular cause of isolated CAKUT in 2.5% of the 590 affected families in this study.

Increased WD-repeat containing protein 1 in interstitial fluid from ovarian carcinomas shown by comparative proteomic analysis of malignant and healthy gynecological tissue.

We aimed to identify differentially expressed proteins in interstitial fluid from ovarian cancer employing multiple fractioning and high resolution mass spectrometry-based proteomic analysis, and asked whether specific proteins that may serve as biomarker candidates or therapeutic targets could be identified. High throughput proteomics was conducted on immunodepleted and fractioned interstitial fluid from pooled samples of ovarian carcinomas, using endometrial carcinomas and healthy ovarian tissue as controls. Differential analysis revealed the up-regulation of extracellular proteasomes in tumor interstitial fluid compared to the healthy control. Moreover, a number of differentially expressed proteins in interstitial fluid from ovarian carcinomas compared with control tissues were identified. Detection of proteasome 20S related proteins in TIF compared to IF from healthy tissue indicates that the 20S proteasome can have a role in the tumor microenvironment. Six selected proteins, CEACAM5, FREM2, MUC5AC, TFF3, PYCARD and WDR1, were independently validated in individual tumor lysates from ovarian carcinomas by multiple reaction monitoring initiated detection and sequence analysis, Western blot and/or selected reaction monitoring. Quantification of specific proteins revealed substantial heterogeneity between individual samples. Nevertheless, WD repeat-containing protein 1 was confirmed as being significantly overexpressed in interstitial fluid from ovarian carcinomas compared to healthy ovarian tissue by Orbitrap analysis of individual native interstitial fluid from ovarian and endometrial carcinomas and healthy ovarian tissue. We suggest that this protein should be explored as a therapeutic target in ovarian carcinomas. This article is part of a Special Issue entitled: An Updated Secretome.