Bone morphogenetic protein 15 supplementation enhances cumulus expansion, nuclear maturation and progesterone production of in vitro-matured bovine cumulus-oocyte complexes.

In vitro embryo production (IVP) efficiency is reduced when compared to in vivo. The basic knowledge of bovine in vitro oocyte maturation (IVM) mechanisms provides support to improve in vitro embryo production yields. The present study assessed the effects of bone morphogenetic protein 15 (BMP15), fibroblast growth factor 16 (FGF16) and their combined action on cumulus cells (CC) expansion, oocyte and CC DNA fragmentation, oocyte nuclear maturation, energetic metabolism and progesterone production in bovine IVM. Cumulus-oocyte complexes (COC) were matured in control or supplemented media containing BMP15 (100 ng/ml), FGF16 (10 ng/ml) or BMP15 combined with FGF16; and assessed at 0 and 22 hr of IVM. BMP15 alone or its association with FGF16 enhanced cumulus expansion. BMP15 decreased DNA fragmentation in both CC and oocytes, and improved oocyte nuclear maturation rate. In addition, BMP15 increased CC progesterone production, an effect not previously reported. The present study reinforces previous data pointing to a beneficial influence of BMP15 during IVM, while providing novel evidence that the underlying mechanisms involve increased progesterone production.

Fibroblast growth factor 16 stimulates proliferation but blocks differentiation of rat stem Leydig cells during regeneration.

OBJECTIVES: We aim to investigate the effects of fibroblast growth factor 16 (FGF16) on Leydig cell regeneration in ethane dimethane sulphonate (EDS)-treated rat testis. METHODS: We intraperitoneally inject 75 mg/kg EDS to adult male Sprague Dawley rats and then intratesticularly inject FGF16 (0, 10 and 100 ng/testis/day) from post-EDS day 14 for 14 days. We investigate serum hormone levels, Leydig cell number, gene and protein expression in vivo. We also explore the effects of FGF16 treatment on stem Leydig cell proliferation in vitro. RESULTS: FGF16 lowers serum testosterone levels (21.6% of the control at a dose of 100 ng/testis) without affecting the levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) on post-EDS day 28 in vivo. FGF16 increases Leydig cell number at doses of 10 and 100 ng/mg without affecting Sertoli cell number, increases the percentage of PCNA-positive Leydig cells, and down-regulates the expression of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Cyp17a1 and Hsd17b3) and Sertoli cell genes (Fshr, Dhh and Sox9) and their proteins in vivo. FGF16 increases phosphorylation of AKT1 and AKT2 as well as EKR1/2 in vivo, indicating that it possibly acts via AKT1/ATK2 and ERK1/2 pathways. FGF16 also lowers medium testosterone levels and down-regulates the expression of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Cyp17a1 and Hsd17b3) but increases EdU incorporation into stem Leydig cells in vitro. CONCLUSIONS: These data suggest that FGF16 stimulates stem and progenitor Leydig cell proliferation but blocks their differentiation, thus lowering testosterone biosynthesis.

GATA4 regulates Fgf16 to promote heart repair after injury.

Although the mammalian heart can regenerate during the neonatal stage, this endogenous regenerative capacity is lost with age. Importantly, replication of cardiomyocytes has been found to be the key mechanism responsible for neonatal cardiac regeneration. Unraveling the transcriptional regulatory network for inducing cardiomyocyte replication will, therefore, be crucial for the development of novel therapies to drive cardiac repair after injury. Here, we investigated whether the key cardiac transcription factor GATA4 is required for neonatal mouse heart regeneration. Using the neonatal mouse heart cryoinjury and apical resection models with an inducible loss of GATA4 specifically in cardiomyocytes, we found severely depressed ventricular function in the Gata4-ablated mice (mutant) after injury. This was accompanied by reduced cardiomyocyte replication. In addition, the mutant hearts displayed impaired coronary angiogenesis and increased hypertrophy and fibrosis after injury. Mechanistically, we found that the paracrine factor FGF16 was significantly reduced in the mutant hearts after injury compared with littermate controls and was directly regulated by GATA4. Cardiac-specific overexpression of FGF16 via adeno-associated virus subtype 9 (AAV9) in the mutant hearts partially rescued the cryoinjury-induced cardiac hypertrophy, promoted cardiomyocyte replication and improved heart function after injury. Altogether, our data demonstrate that GATA4 is required for neonatal heart regeneration through regulation of Fgf16, suggesting that paracrine factors could be of potential use in promoting myocardial repair.

Secretome Screening Reveals Fibroblast Growth Factors as Novel Inhibitors of Viral Replication.

Cellular antiviral programs can efficiently inhibit viral infection. These programs are often initiated through signaling cascades induced by secreted proteins, such as type I interferons, interleukin-6 (IL-6), or tumor necrosis factor alpha (TNF-α). In the present study, we generated an arrayed library of 756 human secreted proteins to perform a secretome screen focused on the discovery of novel modulators of viral entry and/or replication. The individual secreted proteins were tested for the capacity to inhibit infection by two replication-competent recombinant vesicular stomatitis viruses (VSVs) with distinct glycoproteins utilizing different entry pathways. Fibroblast growth factor 16 (FGF16) was identified and confirmed as the most prominent novel inhibitor of both VSVs and therefore of viral replication, not entry. Importantly, an antiviral interferon signature was completely absent in FGF16-treated cells. Nevertheless, the antiviral effect of FGF16 is broad, as it was evident on multiple cell types and also on infection by coxsackievirus. In addition, other members of the FGF family also inhibited viral infection. Thus, our unbiased secretome screen revealed a novel protein family capable of inducing a cellular antiviral state. This previously unappreciated role of the FGF family may have implications for the development of new antivirals and the efficacy of oncolytic virus therapy.IMPORTANCE Viruses infect human cells in order to replicate, while human cells aim to resist infection. Several cellular antiviral programs have therefore evolved to resist infection. Knowledge of these programs is essential for the design of antiviral therapeutics in the future. The induction of antiviral programs is often initiated by secreted proteins, such as interferons. We hypothesized that other secreted proteins may also promote resistance to viral infection. Thus, we tested 756 human secreted proteins for the capacity to inhibit two pseudotypes of vesicular stomatitis virus (VSV). In this secretome screen on viral infection, we identified fibroblast growth factor 16 (FGF16) as a novel antiviral against multiple VSV pseudotypes as well as coxsackievirus. Subsequent testing of other FGF family members revealed that FGF signaling generally inhibits viral infection. This finding may lead to the development of new antivirals and may also be applicable for enhancing oncolytic virus therapy.

Further evidence for FGF16 truncating mutations as the cause of X-linked recessive fusion of metacarpals 4 / 5.

BACKGROUND: Metacarpal 4-5 fusion (MF4; MIM#309630) is a rare congenital malformation of the hand characterized by the partial or complete fusion of the fourth and fifth metacarpals. The anomaly occurs as an isolated trait or part of a genetic syndrome. Recently, we have identified FGF16 nonsense mutations as the underlying cause of isolated X-linked recessive MF4. METHODS: In this report, we provide a detailed clinical description of a sporadic male patient showing MF4 in whom we performed Sanger sequencing of the entire coding sequence of FGF16. RESULTS: In addition to MF4 symptoms, the patient presented with generalized joint laxity and hypermobility. FGF16 sequencing detected a novel truncating mutation (c.474_477del; p.E158DfsX25) in exon 3 of the gene. A heterozygous mutation was found in a clinically and radiologically unaffected mother of the proband. CONCLUSION: Our finding confirms that truncating mutations of FGF16 are causative for X-linked recessive metacarpal 4-5 fusion. Importantly, the mutation detected in this study was located in last exon of the gene (exon 3), like the only two FGF16 disease-causing variants identified to date. Thus, all FGF16 mutations known to give rise to this rare skeletal hand malformation are C-terminal and most probably do not result in a nonsense mediated decay. Additionally, our proband showed mild symptoms of a connective tissue disorder, as some other patients previously reported to have X-linked MF4. Therefore, we suggest that impaired FGF16 function may also be responsible for connective tissue symptoms in MF4 patients.

Whole exome sequencing identifies FGF16 nonsense mutations as the cause of X-linked recessive metacarpal 4/5 fusion.

BACKGROUND: Metacarpal 4-5 fusion (MF4; MIM %309630) is a rare congenital malformation of the hand characterised by the partial or complete fusion of the fourth and fifth metacarpals. The anomaly occurs as an isolated trait or part of a genetic syndrome. METHODS: To search for disease-causing mutation, whole exome sequencing (WES) was performed on samples from a single trio. Before WES, molecular screening including gene sequencing and array comparative genomic hybridisation was applied. Validation of WES and segregation studies were done using routine Sanger sequencing. RESULTS: Exome sequencing detected a nonsense mutation (c.C535T; p.R179X) in exon 3 of the FGF16 gene, which maps to chromosome Xq21.1. Mutational screening of the FGF16 gene performed in an unrelated proband of different ethnicity showed another nonsense mutation in exon 3 (c.C470A; p.S157X). CONCLUSIONS: This study shows that truncating mutations of FGF16 are associated with X-linked recessive metacarpal 4-5 fusion. The study provides evidence for the involvement of FGF16 in the fine tuning of the human skeleton of the hand.

Reprogramming of glucose metabolism via PFKFB4 is critical in FGF16-driven invasion of breast cancer cells.

Fibroblast growth factors (FGFs) are expressed in both developing and adult tissues and play important roles in embryogenesis, tissue homeostasis, angiogenesis, and neoplastic transformation. Here, we report the elevated expression of FGF16 in human breast tumor and investigate its potential involvement in breast cancer progression. The onset of epithelial-mesenchymal transition (EMT), a prerequisite for cancer metastasis, was observed in human mammary epithelial cell-line MCF10A by FGF16. Further study unveiled that FGF16 alters mRNA expression of a set of extracellular matrix genes to promote cellular invasion. Cancer cells undergoing EMT often show metabolic alteration to sustain their continuous proliferation and energy-intensive migration. Similarly, FGF16 induced a significant metabolic shift toward aerobic glycolysis. At the molecular level, FGF16 enhanced GLUT3 expression to facilitate glucose transport into cells, which through aerobic glycolysis generates lactate. The bi-functional protein, 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 4 (PFKFB4) was found to be a mediator in FGF16-driven glycolysis and subsequent invasion. Furthermore, PFKFB4 was found to play a critical role in promoting lactate-induced cell invasion since silencing PFKFB4 decreased lactate level and rendered the cells less invasive. These findings support potential clinical intervention of any of the members of FGF16-GLUT3-PFKFB4 axis to control the invasion of breast cancer cells.

Patient-specific iPSC-derived cardiomyocytes reveal abnormal regulation of FGF16 in a familial atrial septal defect.

AIMS: Congenital heart disease (CHD) frequently occurs in newborns due to abnormal formation of the heart or major blood vessels. Mutations in the GATA4 gene, which encodes GATA binding protein 4, are responsible for atrial septal defect (ASD), a common CHD. This study aims to gain insights into the molecular mechanisms of CHD using human-induced pluripotent stem cells (iPSCs) from a family cohort with ASD. METHODS AND RESULTS: Patient-specific iPSCs possess the same genetic information as the donor and can differentiate into various cell types from all three germ layers in vitro, thus presenting a promising approach for disease modelling and molecular mechanism research. Here, we generated a patient-specific iPSC line (iPSC-G4T280M) from a family cohort carrying a hereditary ASD mutation in GATA4 gene (T280M), as well as a human embryonic stem cell line (ESC-G4T280M) carrying the isogenic T280M mutation using the CRISPR/Cas9 genome editing method. The GATA4-mutant iPSCs and ESCs were then differentiated into cardiomyocytes (CMs) to model GATA4 mutation-associated ASD. We observed an obvious defect in cell proliferation in cardiomyocytes derived from both GATA4T280M-mutant iPSCs (iPSC-G4T280M-CMs) and ESCs (ESC-G4T280M-CMs), while the impaired proliferation ability of iPSC-G4T280M-CMs could be restored by gene correction. Integrated analysis of RNA-Seq and ChIP-Seq data indicated that FGF16 is a direct target of wild-type GATA4. However, the T280M mutation obstructed GATA4 occupancy at the FGF16 promoter region, leading to impaired activation of FGF16 transcription. Overexpression of FGF16 in GATA4-mutant cardiomyocytes rescued the cell proliferation defect. The direct relationship between GATA4T280M and ASD was demonstrated in a human iPSC model for the first time. CONCLUSIONS: In summary, our study revealed the molecular mechanism of the GATA4T280M mutation in ASD. Understanding the roles of the GATA4-FGF16 axis in iPSC-CMs will shed light on heart development and provide novel insights for the treatment of ASD and other CHD disorders.

FGF16 regulated by miR-520b enhances the cell proliferation of lung cancer.

FGF16 is implicated in the progression of some specific types of cancers, such as embryonic carcinoma, ovarian cancer, and liver cancer. Yet, the function of FGF16 in the development of lung cancer remains largely unexplored. In this study, we present the novel function of FGF16 and the regulation of miR-520b on FGF16 in lung cancer progression. In clinical lung cancer tissues, FGF16 is overexpressed and its high level is negatively associated with the low level of miR-520b. Furthermore, both the transcription and translation levels of FGF16 are restrained by miR-520b in lung cancer cells. For the regulatory mechanism investigation, miR-520b is able to directly bind to the 3'-untranslated region (3'UTR) of FGF16 mRNA, leading to its mRNA cleavage in the cells. Functionally, miR-520b reduces the growth of lung cancer and its inhibitor anti-miR520b is able to promote the growth through competing endogenous miR-520b. Moreover, FGF16 silence using RNA interference is capable of doing great damage to anti-miR-520b-accelerated growth of lung cancer. Thus, our finding indicates that FGF16 is a new target gene of miR-520b in lung cancer. For lung cancer, FGF16 may serve as a novel biomarker and miR-520b/FGF16 may be useful in clinical treatment.

MiRNA-144-3p inhibits high glucose induced cell proliferation through suppressing FGF16.

As a major cause of blindness, diabetic retinopathy (DR) is often found in the developed countries. Our previous study identified a down-regulated miRNA: miR-144-3p in response to hyperglycemia. The present study aims to investigate the role of miR-144-3p in proliferation of microvascular epithelial cells. Endothelial cells were treated with different concentrations of glucose, after which miR-144-3p were detected with real-time PCR assay. MiR-144-3p mimics or inhibitors were used to increase or knockdown the level of this miRNA. Western blotting assay and ELISA assay were used to measure the expression and concentration of VEGF protein. 5-Bromo-2-deoxyUridine (BrdU) labeled cell cycle assay was used to detect cells in S phase. MiRNA targets were predicted by using a TargetScan tool, and were further verified by luciferase reporter assay. In the present study, we focussed on a significantly down-regulated miRNA, miR-144-3p, and investigated its role in high glucose (HG) induced cell proliferation. Our data showed that miR-144-3p mimics significantly inhibited HG induced cell proliferation and reduced the percentage of cells in S phase. HG induced up-regulation of VEGF was also prohibited by miR-144-3p mimics. Through wound-healing assay, we found that miR-144-3p suppressed cell migration after HG treatments. Moreover, we predicted and proved that fibroblast growth factor (FGF)16 is a direct target of miR-144-3p. Finally, miR-144-3p attenuated HG induced MAPK activation. In conclusion, we demonstrated that miR-144-3p inhibited high glucose-induced cell proliferation through suppressing FGF16 and MAPK signaling pathway, suggesting a possible role of miR-144-FGF16 in the development of DR.

Cardiac Fgf-16 Expression Supports Cardiomyocyte Survival and Increases Resistance to Doxorubicin Cytotoxicity.

The fibroblast growth factor (FGF) 16 gene is preferentially expressed by cardiomyocytes after birth with levels increasing into adulthood. Null mice and isolated heart studies suggest a role for FGF-16 in cardiac maintenance and survival, including increased resistance to doxorubicin (DOX)-induced injury. A single treatment with DOX was also shown to rapidly deplete endogenous rat FGF-16 mRNA at 6 h in both adult heart and neonatal cardiomyocytes. However, the effect of DOX on rat cardiac function at the time of decreased FGF-16 gene expression and the effect of FGF-16 availability on cardiomyocyte survival, including in the context of acute DOX cytotoxicity, have not been reported. The objective was to assess the effect of acute (6 and 24 h) DOX treatment on cardiac function and the effects of FGF-16 small interfering RNA "knockdown," as well as adenoviral overexpression, in the context of acute DOX cytotoxicity, including cardiomyocyte survival and DOX efflux transport. A significant decrease in heart systolic function was detected by echocardiography in adult rats treated with 15 mg DOX/kg at 6 h; however, unlike FGF-16, there was no change in atrial natriuretic peptide transcript levels. Both systolic and diastolic dysfunctions were observed at 24 h. In addition, specific FGF-16 "knockdown" in neonatal rat cardiomyocytes results in a significant increase in cell death. Conversely, adenoviral FGF-16 overexpression was associated with a significant decrease in cardiomyocyte injury as a result of 1 µM DOX treatment. A specific increase in efflux transporter gene expression and DOX efflux was also seen, which is consistent with a reduction in DOX cytotoxicity. Finally, the increased efflux and decreased DOX-induced damage with FGF-16 overexpression were blunted by inhibition of FGF receptor signaling. These observations are consistent with FGF-16 serving as an endogenous cardiomyocyte survival factor, which may involve a positive effect on regulating efflux transport to reduce cardiotoxicity.

MiR-520f promotes cell aggressiveness by regulating fibroblast growth factor 16 in hepatocellular carcinoma.

Cancer metastasis is a multistep cellular process, which has be confirmed one of mainly causes of cancer associated-death in hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) participate in tumorigenesis function as either tumor suppressor genes or oncogenes. In order to elaborate the critical miRNAs and their targets in HCC, we compared the differential expression of miRNA between HCC tissues and normal tissues. Microarray analysis revealed there were several significantly up-expression miRNAs in HCC, compared to normal solid tissue. Among them, the expression of miR-520f was the most over-expression in HCC cell lines than that in human normal liver cells LO2, as well as up-regulated in HCC than that in the corresponding normal tissues. Moreover, Kaplan Meier-plotter analyses revealed that higher miR-520f levels were negatively correlated with poor overall survival. By applying bioinformatics methods to identify the targeting genes of miRNA, we demonstrated that fibroblast growth factor 16 (FGF16) was the miR-520f-targeted gene. Meanwhile, FGF16 exhibited similar expression patterns to miR-520f in HCC. Forced miR-520f expression accelerated HCC cells proliferation and aggressiveness in vitro and in vivo, whereas down-regulation of miR-520f caused an opposite outcome. Moreover, over-expression of FGF16 was closely related to the metastatic potential of HCC cells. Herein, we also confirmed that ectopic expression of FGF16 in HCC cells promoted proliferation, colony formation, and increased migration, invasion of HCC cells in vitro. Collectively, our results indicated that over-expression of miR-520f and FGF16 was positively associated with aggressive phenotypes and poor survival of patients with HCC, and miR-520f promoted HCC aggressive phenotypes by regulating the expression of FGF16. MiR-520f may be employed as a prognostic factor and therapeutic target for HCC.

FGF-16 protects against adverse cardiac remodeling in the infarct diabetic heart.

Till now, no functional studies for FGF-16 in diabetic heart have been reported. Therefore, this study aims to evaluate the potential function of FGF-16 in inhibiting adverse cardiac remodeling in post myocardial infarction (MI) of diabetic heart. We investigated the role of fibroblast growth factor-16 (FGF-16) in post-MI remodeling and role of cardio-protection in the diabetic infarct heart. Adult db/db diabetic mice were assigned to sham group, MI group and MI+FGF-16 group, respectively. MI group was induced by permanent coronary artery ligation, and the mice were subjected to 2D trans-thoracic echocardiography 2-4 weeks post-surgery. The results showed that the infiltration of monocytes, the associated pro-inflammatory cytokines were significantly increased, and the adverse cardiac remodeling and left ventricular dysfunction were observed in MI group. FGF-16 treatment protected against apoptosis, cardiac dysfunction and chamber dilatation post-MI, and decreased monocyte infiltration and cardiomyocyte hypertrophy/apoptosis. Meanwhile, the FGF-16 treatment also attenuated interstitial fibrosis and myocardial inflammation post-MI, increased M2 macrophage differentiation and associated anti-inflammatory cytokines, reduced adverse remodeling, and improved cardiac function. In conclusion, our results suggest that the heart appears to be a target of systemic and possibly locally generated FGF-16, which plays a therapeutic role in cardiac protection in the post-MI diabetic heart.

Expression of the Cardiac Maintenance and Survival Factor FGF-16 Gene Is Regulated by Csx/Nkx2.5 and Is an Early Target of Doxorubicin Cardiotoxicity.

The fibroblast growth factor (FGF) 16 gene (Fgf-16) is preferentially expressed by neonatal cardiomyocytes after birth, with levels increasing into adulthood. Null mice and isolated heart studies suggest a role for FGF-16 in cardiac maintenance and survival, including increased resistance to doxorubicin (DOX)-induced injury. However, the effect of DOX on endogenous FGF-16 synthesis and specifically regulation of cardiac Fgf-16 expression has not been reported. Here we assess the effect of DOX on FGF-16 RNA levels and stability as well as promoter activity and use sequence analysis, knockdown, and overexpression to investigate the role of cardiac transcription factor(s) implicated in the response. Endogenous FGF-16 RNA levels were reduced >70% in 8-week-old rats treated with 15 mg DOX/kg for 6 h. This was modeled in neonatal rat cardiomyocyte cultures, where an equivalent decrease was also seen within 6 h of 1 µM DOX treatment. Six kilobases of mouse Fgf-16 upstream flanking and promoter DNA was also assessed for DOX responsiveness in transfected cardiomyocytes. A decrease in FGF-16 promoter activity was seen with only 747 base pairs containing the Fgf-16 TATA box that includes a putative and highly conserved binding site for the cardiac transcription factor Csx/Nkx2.5. There was also no effect of DOX on FGF-16 RNA stability, consistent with transcriptional control. Levels and binding of Csx/Nkx2.5 to the FGF-16 promoter were reduced with DOX treatment. Knockdown of Csx/Nkx2.5 specifically decreased endogenous FGF-16 RNA and protein levels, whereas Csx/Nkx2.5 overexpression stimulated levels, and increased resistance to the rapid DOX-induced depletion of FGF-16. These observations indicate that Fgf-16 expression is directly regulated by Csx/Nkx2.5 in neonatal cardiomyocytes, and a negative effect of DOX on Csx/Nkx2.5 and, thus, endogenous FGF-16 synthesis may contribute indirectly to its cardiotoxic effects. Targeting FGF-16 levels could, however, offer increased resistance to cardiac injury.

Heart-specific expression of FGF-16 and a potential role in postnatal cardioprotection.

Fibroblast growth factor 16 (FGF-16) was originally cloned from rat heart. Subsequent investigation of mouse FGF-16, including generation of null mice, revealed a specific pattern of expression in the endocardium and epicardium, and role for FGF-16 during embryonic heart development. FGF-16 is expressed mainly in brown adipose tissue during rat embryonic development, but is expressed mainly in the murine heart after birth. There is also an apparent switch from limited endocardial and epicardial expression in the embryo to the myocardium in the perinatal period. The FGF-16 gene and its location on the X chromosome are conserved between human and murine species, and no other member of the FGF family shows this pattern of spatial and temporal expression. The human and murine FGF-16 gene promoter regions also share an equivalent location for TATA sequences, as well as adjacent putative binding sites for transcription factors linked to cardiac expression and response to stress. Recent evidence has implicated nonsense mutation of FGF-16 with increased cardiovascular risk, and FGF-16 supplementation with cardioprotection. Here we review the important role of FGF-16 in embryonic heart development, its gene regulation, and evidence for FGF-16 as an endogenous and exogenous cardiac-specific and protective factor in the postnatal heart. Moreover, given the conservation of the FGF-16 gene and its chromosomal location between species, the question of support for a cardiac role in the human population is also considered.

Identification of three novel FGF16 mutations in X-linked recessive fusion of the fourth and fifth metacarpals and possible correlation with heart disease.

Nonsense mutations in FGF16 have recently been linked to X-linked recessive hand malformations with fusion between the fourth and the fifth metacarpals and hypoplasia of the fifth digit (MF4; MIM#309630). The purpose of this study was to perform careful clinical phenotyping and to define molecular mechanisms behind X-linked recessive MF4 in three unrelated families. We performed whole-exome sequencing, and identified three novel mutations in FGF16. The functional impact of FGF16 loss was further studied using morpholino-based suppression of fgf16 in zebrafish. In addition, clinical investigations revealed reduced penetrance and variable expressivity of the MF4 phenotype. Cardiac disorders, including myocardial infarction and atrial fibrillation followed the X-linked FGF16 mutated trait in one large family. Our findings establish that a mutation in exon 1, 2 or 3 of FGF16 results in X-linked recessive MF4 and expand the phenotypic spectrum of FGF16 mutations to include a possible correlation with heart disease.