A cellular model provides insights into the pathogenicity of the oncogenic FOXL2 somatic variant p.Cys134Trp.

BACKGROUND: FOXL2 is a transcription factor expressed in ovarian granulosa cells. A somatic variant of FOXL2 (c.402 C > G, p.Cys134Trp) is the hallmark of adult-type granulosa cell tumours. METHODS: We generated KGN cell clones either heterozygous for this variant (MUT) or homozygous for the wild-type (WT) allele by CRISPR/Cas9 editing. They underwent RNA-Seq and bioinformatics analyses to uncover pathways impacted by deregulated genes. Cell morphology and migration were studied. RESULTS: The differentially expressed genes (DEGs) between WT/MUT and WT/WT KGN cells (DEGs-WT/MUT), pointed to several dysregulated pathways, like TGF-beta pathway, cell adhesion and migration. Consistently, WT/MUT cells were rounder than WT/WT cells and displayed a different distribution of stress fibres and paxillin staining. A comparison of the DEGs-WT/MUT with those found when FOXL2 was knocked down (KD) in WT/WT KGN cells showed that most DEGs-WT/MUT cells were not so in the KD experiment, supporting a gain-of-function (GOF) scenario. MUT-FOXL2 also displayed a stronger interaction with SMAD3. CONCLUSIONS: Our work, aiming at better understanding the GOF scenario, shows that the dysregulated genes and pathways are consistent with this idea. Besides, we propose that GOF might result from an enhanced interaction with SMAD3 that could underlie an ectopic capacity of mutated FOXL2 to bind SMAD4.

The forkhead DNA-binding domain binds specific G2-rich RNA sequences.

Transcription factors contain a DNA-binding domain ensuring specific recognition of DNA target sequences. The family of forkhead (FOX) transcription factors is composed of dozens of paralogs in mammals. The forkhead domain (FHD) is a segment of about 100 amino acids that binds an A-rich DNA sequence. Using DNA and RNA PCR-SELEX, we show that recombinant FOXL2 proteins, either wild-type or carrying the oncogenic variant C134W, recognize similar DNA-binding sites. This suggests that the oncogenic variant does not alter the intrinsic sequence-specificity of FOXL2. Most importantly, we show that FOXL2 binds G2-rich RNA sequences whereas it virtually fails to bind similar sequences in DNA chemistry. Interestingly, a statistically significant subset of genes responding to the knock-down of FOXL2/Foxl2 harbor such G2-rich sequences and are involved in crucial signaling pathways and cellular processes. In addition, we show that FOXA1, FOXO3a and chimeric FOXL2 proteins containing the FHD of the former are also able to interact with some of the preferred FOXL2-binding sequences. Our results point to an unexpected and novel characteristic of the forkhead domain, the biological relevance of which remains to be explored.

Dominant negative variants and cotranslational assembly of macromolecular complexes.

Pathogenic variants occurring in protein-coding regions underlie human genetic disease through various mechanisms. They can lead to a loss of function (LOF) such as in recessive conditions or in dominant conditions due to haploinsufficiency. Dominant-negative (DN) effects, counteracting the activity of the normal gene-product, and gain of function (GOF) are also mechanisms driving dominance. Here, I discuss a few papers on these specific mechanisms. In short, there is accumulating evidence pointing to differences between LOF versus non-LOF variants (DN and GOF). The latter are thought to have milder effects on protein structure and, as expected, DN variants are enriched at protein interfaces. This tendency to cluster in 3D space can help improve the ability of computational tools to predict the pathogenicity of DN variants, which is currently a challenging issue. More recent results support the hypothesis whereby cotranslational assembly of macromolecular complexes can buffer deleterious consequences of variants that would otherwise lead to DN effects (DNEs). Indeed, subunits the variants of which are responsible for DNEs tend to elude cotranslational assembly, thus poisoning complexes involving wild-type subunits. The constraints explaining why the buffering of DNEs is not universal require further investigation.

The Oncogenic FOXL2 C134W Mutation Is a Key Driver of Granulosa Cell Tumors.

Adult-type granulosa cell tumors (AGCT) are the most common type of malignant ovarian sex cord-stromal tumors. Most AGCTs carry the somatic variant c.402C>G (p.C134W) affecting the transcription factor FOXL2. Germline dominant variants in FOXL2 are responsible for blepharophimosis syndrome, which is characterized by underdevelopment of the eyelid. In this work, we generated a mouse model harboring the C134W variant of FOXL2 to evaluate in vivo the poorly understood oncogenic role of FOXL2. The mutation was dominant regarding eyelid hypoplasia, reminiscent of blepharophimosis syndrome. Interestingly, Foxl2+/C134W female mice had reduced fertility and developed AGCTs through a progression from abnormal ovaries with aberrant granulosa cells to ovaries with stromal hyperplasia and atypia and on to tumors in adut mice. The genes dysregulated in mouse AGCTs exhibited the hallmarks of cancer and were consistent with a gain-of-function of the mutated allele affecting TGFß signaling. A comparison of these data with previous results on human AGCTs indicated similar deregulated pathways. Finally, a mutational analysis of mouse AGCT transcriptomic data suggested the absence of additional driver mutations apart from FOXL2-C134W. These results provide a clear in vivo example in which a single mutational hit triggers tumor development associated with profound transcriptomic alterations. SIGNIFICANCE: A newly generated mouse model carrying a FOXL2 mutation characteristic of adult-type granulosa cell tumors shows that FOXL2 C134W shifts the transcriptome towards a signature of granulosa cell cancer and drives tumorigenesis.

Recurrent missense variants in clonal hematopoiesis-related genes present in the general population.

Clonal hematopoiesis (CH) consists in an abnormal expansion of a hematopoietic stem cell bearing an advantageous somatic variant. A survey of known recurrent somatic missense variants in DNMT3A, SF3B1, SRSF2, and TP53, some of the most prominent genes underlying CH of indeterminate potential (CHIP), in gnomAD noncancer database shows the presence of 73 variants. Many of them reach frequencies higher than 0.01% in various populations and, in many cases, are enriched in specific populations. Consistent with a potential involvement in CHIP, we found that the age distribution of the carriers is shifted towards old ages. Moreover, the variant allele frequencies are on average lower than 50%, expected for germline heterozygous variants. The pervasive presence of some of such variants in blood DNA from elder individuals is compatible with CHIP of somatic origin. On practical grounds, CHIP can lead to misclassification of somatic variants in cancer-predisposition genes as inherited, which bear consequences for the affected individuals and their families.

A truncating variant of RAD51B associated with primary ovarian insufficiency provides insights into its meiotic and somatic functions.

Primary ovarian insufficiency (POI) causes female infertility by abolishing normal ovarian function. Although its genetic etiology has been extensively investigated, most POI cases remain unexplained. Using whole-exome sequencing, we identified a homozygous variant in RAD51B -(c.92delT) in two sisters with POI. In vitro studies revealed that this variant leads to translation reinitiation at methionine 64. Here, we show that this is a pathogenic hypomorphic variant in a mouse model. Rad51bc.92delT/c.92delT mice exhibited meiotic DNA repair defects due to RAD51 and HSF2BP/BMRE1 accumulation in the chromosome axes leading to a reduction in the number of crossovers. Interestingly, the interaction of RAD51B-c.92delT with RAD51C and with its newly identified interactors RAD51 and HELQ was abrogated or diminished. Repair of mitomycin-C-induced chromosomal aberrations was impaired in RAD51B/Rad51b-c.92delT human and mouse somatic cells in vitro and in explanted mouse bone marrow cells. Accordingly, Rad51b-c.92delT variant reduced replication fork progression of patient-derived lymphoblastoid cell lines and pluripotent reprogramming efficiency of primary mouse embryonic fibroblasts. Finally, Rad51bc.92delT/c.92delT mice displayed increased incidence of pituitary gland hyperplasia. These results provide new mechanistic insights into the role of RAD51B not only in meiosis but in the maintenance of somatic genome stability.

Who ever thought genetic mutations were random?

In a recent study of de novo mutations in arabidopsis (Arabidopsis thaliana), Monroe et al. found a lower mutation frequency inside gene bodies and certain essential genes, shattering the long-standing idea that mutations are entirely random across the genome. Here I highlight the molecular nonrandomness of mutations and the type of gene affected.

Pathogenic "germline" variants associated with myeloproliferative disorders in apparently normal individuals: Inherited or acquired genetic alterations?

Myeloproliferative syndromes (MPS) are hematologic malignancies due to the expansion of an abnormal hematopoietic stem cell. They include chronic myeloid leukemia (CML) and non-CML MPS such as polycythemia vera, essential thrombocythemia and primary myelofibrosis. The latter are distinguished by somatic pathogenic variants affecting JAK2, CALR, or MPL genes. Apparent germline pathogenic variants have been reported in the general population. Here, we found that two gnomAD data-sets report more homozygotes than expected for the JAK2 c.1849G > T(Val617Phe) variant. We propose that somatic gene conversion can explain the presence of those unexpected homozygotes in normal populations. Consistently, homozygous individuals are older than 65 years. We also found a lower-than-expected frequency of the JAK2 variant in younger individuals suggesting that somatic mutation can underlie its presence in (at least some) heterozygotes. Regarding pathogenic variants in MPL and CALR, they are also present in the gnomAD data-sets explored. However, we cannot conclude that such seemingly germline variants are in fact somatic alterations. These results suggest that apparently normal individuals bearing MPS-related variants can be subclinical/undiagnosed MPS cases of somatic origin. It would be interesting to assess the hematologic phenotype of such individuals and the presence of the relevant variants in other tissues.

Gene-dosage issues: a recurrent theme in whole genome duplication events.

Two recent studies have addressed the long-term consequences of whole genome duplications (WGD). Specifically, they analyzed transcriptomes of the plant Arabidopsis thaliana and of four salmonids to assess the impact of WGD on gene expression. These studies point to commonalities in gene expression adjustments after polyploidization that we outline and discuss below.

One Hundred Years of Gene Balance: How Stoichiometric Issues Affect Gene Expression, Genome Evolution, and Quantitative Traits.

A century ago experiments with the flowering plant Datura stramonium and the fruit fly Drosophila melanogaster revealed that adding an extra chromosome to a karyotype was much more detrimental than adding a whole set of chromosomes. This phenomenon was referred to as gene balance and has been recapitulated across eukaryotic species. Here, we retrace some developments in this field. Molecular studies suggest that the basis of balance involves stoichiometric relationships of multi-component interactions. This concept has implication for the mechanisms controlling gene expression, genome evolution, sex chromosome evolution/dosage compensation, speciation mechanisms, and the underlying genetics of quantitative traits.

FOXL2 in adult-type granulosa cell tumour of the ovary: oncogene or tumour suppressor gene?

A recurrent mutation in FOXL2 (c.402C>G; p.C134W) is present in over 95% of adult-type granulosa cell tumours (AGCTs). In contrast, various loss-of-function mutations in FOXL2 lead to the development of blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES). BPES is characterised by an eyelid malformation often accompanied with primary ovarian insufficiency. Two recent studies suggest that FOXL2 C402G is a gain- or change-of-function mutation with altered DNA-binding specificity. Another study proposes that FOXL2 C402G is selectively targeted for degradation, inducing somatic haploinsufficiency, suggesting its role as a tumour suppressor. The latter study relies on data indicative of an FOXL2 allelic imbalance in AGCTs. Here we present RNA-seq data as genetic evidence that no real allelic imbalance is observed at the transcriptomic level in AGCTs. Additionally, there is no loss of protein expression in tumours harbouring the mutated allele. These data and other features of this mutation compared to other oncogenes and tumour suppressor genes argue strongly against FOXL2 being a tumour suppressor in this context. Given the likelihood that FOXL2 C402G is oncogenic, targeting the variant protein or its downstream consequences is the most viable path forward to identifying an effective treatment for this cancer. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Genes Encoding Teleost Orthologs of Human Haploinsufficient and Monoallelically Expressed Genes Remain in Duplicate More Frequently Than the Whole Genome.

Gene dosage is an important issue both in cell and evolutionary biology. Most genes are present in two copies or alleles in diploid eukariotic cells. The most outstanding exception is monoallelic gene expression (MA) that concerns genes localized on the X chromosome or in regions undergoing parental imprinting in eutherians, and many other genes scattered throughout the genome. In diploids, haploinsufficiency (HI) implies that a single functional copy of a gene in a diploid organism is insufficient to ensure a normal biological function. One of the most important mechanisms ensuring functional innovation during evolution is whole genome duplication (WGD). In addition to the two WGDs that have occurred in vertebrate genomes, the teleost genomes underwent an additional WGD, after their divergence from tetrapods. In the present work, we have studied on 57 teleost species whether the orthologs of human MA or HI genes remain more frequently in duplicates or returned more frequently in singleton than the rest of the genome. Our results show that the teleost orthologs of HI human genes remained more frequently in duplicate than the rest of the genome in all of the teleost species studied. No signal was observed for the orthologs of genes mapping to the human X chromosome or subjected to parental imprinting. Surprisingly, the teleost orthologs of the other human MA genes remained in duplicate more frequently than the rest of the genome for most teleost species. These results suggest that the teleost orthologs of MA and HI human genes also undergo selective pressures either related to absolute protein amounts and/or of dosage balance issues. However, these constraints seem to be different for MA genes in teleost in comparison with human genomes.

Genomic exploration of the targets of FOXL2 and ESR2 unveils their implication in cell migration, invasion, and adhesion.

FOXL2 and ESR2 are key transcriptional regulators in ovarian granulosa cells. To explore their transcriptional roles and their interplay, we have depleted Foxl2 and Esr2 in mouse primary granulosa cells to assess their ability to bind their targets and/or to modulate gene expression and cellular functions. We show that FOXL2 is involved in a large number of regulatory actions essential for the maintenance of granulosa cell fate. A parallel ChIP-seq analysis showed that FOXL2 mainly binds to sites located in intergenic regions quite far from its targets. A bioinformatic analysis demonstrated that FOXL2-activated genes were enriched in peaks associated with the H3K27ac mark, whereas FOXL2-repressed genes were not, suggesting that FOXL2 can activate transcription through binding to enhancer sites. We also identified about 500 deregulated genes upon Esr2 silencing, of which one third are also targets of FOXL2. We provide evidence showing that both factors modulate, through a coherent feed-forward loop, a number of common targets. Many of the FOXL2/ESR2 targets are involved in cell motility and, consistently, granulosa cells depleted for either Foxl2 or Esr2 exhibit decreased migration, invasion and adhesion. This effect is paralleled by the depletion of their target Phactr1, involved in actin cytoskeleton dynamics. Our analysis expands the number of direct and indirect transcriptional targets of both FOXL2 and ESR2, which deserve investigation in the context of adult-type granulosa cell tumors whose molecular diagnostic hallmark is the presence of the C134W FOXL2 pathogenic variant.

Insights into the pathogenicity of missense variants in the forkhead domain of FOX proteins underlying Mendelian disorders.

Forkhead box (FOX) proteins are members of a conserved family of transcription factors. Pathogenic variants in FOX genes have been shown to be responsible for several human genetic diseases. Here, we have studied the molecular and structural features of germline pathogenic variants in seven FOX proteins involved in Mendelian disorders and compared them with those of variants present in the general population (gnomAD). Our study shows that the DNA-binding domain of FOX proteins is particularly sensitive to damaging variation, although some family members show greater mutational tolerance than others. Next, we set to demonstrate that this tolerance depends on the inheritance mode of FOX-linked disorders. Accordingly, genes whose variants underlie recessive conditions are supposed to have a greater tolerance to variation. This is what we found. As expected, variants responsible for disorders with a dominant inheritance pattern show a higher degree of pathogenicity compared to those segregating in the general population. Moreover, we show that pathogenic and likely pathogenic variants tend to affect mutually exclusive sites with respect to those reported in gnomAD. The former also tend to affect sites with lower solvent exposure and a higher degree of conservation. Our results show the value of using publicly available databases and bioinformatics to gain insights into the molecular and structural bases of disease-causing genetic variation.

Predictable increase in female reproductive window: A simple model connecting age of reproduction, menopause, and longevity.

With the ever-increasing lifespan along with societal changes, women can marry and procreate later than in previous centuries. However, pathogenic genetic variants segregating in the population can lead to female subfertility or infertility well before the average age of normal menopause, leading to counter-selection of such deleterious alleles. In reviewing this field, we speculate that a logical consequence would be the later occurrence of menopause and the extension of women's reproductive lifespan. We illustrate this point with a simple model that applies to other variants that contribute to female infertility, including epigenetic variation. We also consider the effect of medical interventions and lifestyle.

Forkhead Transcription Factors in Health and Disease.

Forkhead box (FOX) proteins belong to an evolutionarily conserved family of transcription factors that has evolved by gene/genome duplication. FOX family members have undergone sequence and regulatory diversification. However, they have retained some degree of functional redundancy, in addition to playing specific roles, both during development and in the adult. Genetic alterations or misregulation of FOX genes underlie human genetic diseases, cancer, and/or aging. In this review, we provide an updated overview of the main characteristics of the members of this family, in terms of breadth of expression, protein domain composition, evolution, and function.

A missense in HSF2BP causing primary ovarian insufficiency affects meiotic recombination by its novel interactor C19ORF57/BRME1.

Primary Ovarian Insufficiency (POI) is a major cause of infertility, but its etiology remains poorly understood. Using whole-exome sequencing in a family with three cases of POI, we identified the candidate missense variant S167L in HSF2BP, an essential meiotic gene. Functional analysis of the HSF2BP-S167L variant in mouse showed that it behaves as a hypomorphic allele compared to a new loss-of-function (knock-out) mouse model. Hsf2bpS167L/S167L females show reduced fertility with smaller litter sizes. To obtain mechanistic insights, we identified C19ORF57/BRME1 as a strong interactor and stabilizer of HSF2BP and showed that the BRME1/HSF2BP protein complex co-immunoprecipitates with BRCA2, RAD51, RPA and PALB2. Meiocytes bearing the HSF2BP-S167L variant showed a strongly decreased staining of both HSF2BP and BRME1 at the recombination nodules and a reduced number of the foci formed by the recombinases RAD51/DMC1, thus leading to a lower frequency of crossovers. Our results provide insights into the molecular mechanism of HSF2BP-S167L in human ovarian insufficiency and sub(in)fertility.