A Progeroid Syndrome Caused by RAF1 deficiency Underscores the importance of RTK signaling for Human Development

This article has been Reviewed by the following groups

Read the full article See related articles

Listed in

Log in to save this article


Somatic and germline gain-of-function point mutations in RAF, the first oncogene to be discovered in humans, delineate a group of tumor-prone syndromes known as RASopathies. In this study, we document the first human phenotype resulting from the germline loss of function of the proto-oncogene RAF1 ( a.k.a. CRAF) . In a consanguineous family, we uncovered a homozygous p.Thr543Met mutation segregating with a neonatal lethal progeroid syndrome with cutaneous, craniofacial, cardiac and limb anomalies. Structure-based prediction and functional tests using human knock-in cells showed that threonine 543 is essential to: 1) ensure RAF1’s stability and phosphorylation, 2) maintain its kinase activity towards substrates of the MAPK pathway and 3) protect from stress-induced apoptosis. When injected in Xenopus embryos mutant RAF1 T543M failed to phenocopy the effects of overactive FGF/MAPK signaling confirming its hypomorphic activity. Collectively, our data disclose the genetic and molecular etiology of a novel segmental progeroid syndrome which highlights the importance of RTK signaling for human development and homeostasis.

Short summary

A germline homozygous recessive loss-of-function mutation p.T453M in RAF1 causes a neonatal lethal progeroid syndrome. In vitro and in vivo tests demonstrate that Thr543 is necessary for RAF1’s protein stability, to transduce signaling to the MAPK pathway and to respond to stress-induced apoptosis.

Article activity feed

  1. Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    the authors do not wish to provide a response at this time.

  2. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    The manuscript by Wong et al., provides the first report of a homozygous loss-of-function mutation of the RAF1 gene in humans. The mutation (T543M) was found in two siblings of a consanguineous family in association with perinatal death and multiple developmental abnormalities. These abnormalities show strong similarities with a rare congenital malformation syndrome with unknown aetiology named Acro-cardio-facial syndrome (ACFS, MIM600460). Conversely, reported abnormalities are different from those observed in RASopathies, congenital diseases caused by gain-of-function mutations in either the RAF gene or genes implicated in the same signaling pathway (MAPK) and that induce its ectopic/over-activation. By performing functional experiments in cellular systems (cell line where the mutated RAF was either overexpressed or knocked-in) and in Xenopus Laevis embryos, the authors demonstrate that the reason for the phenotypic differences compared to RASopathies is that the RAF1T543M variant impairs the signaling activity of RAF and blunts MAPK pathway activation. In particular, the RAF1T543M variant: 1) is not actively phosphorylated at key activating residues, 2) is unable to transduce MAPK signaling towards MEK/ERK substrates, 3) is inherently unstable and prone to proteasome-mediated degradation and 4) is unable to block stress-induced apoptosis. On the basis of increased apoptosis detected in cellular systems and some morphological defects observed in the probands, the author classify this novel syndrome as a segmental progeroid syndrome.

    Major comments:

    The authors perform a thorough analysis of the structural and functional defects of the RAF1T543M protein, using in silico analyses and in vitro systems. The data are corroborated by an elegant and clear-cut experiment in Xenopus embryos that demonstrates that the mutated RAF is not able to transduce MAPK signaling when overexpressed in an in vivo model. The lack of patients' material prevents a validation in human cells, but I think the evidence collected in the manuscript is supportive of the loss-of-function mutation in RAF as the causative mutation of the observed phenotype.

    The concept illustrated in the last sentence of the discussion, i.e. that different mutations in the same genes can either cause cancer (when overactivating) or premature aging (when blunting the activity of the enzyme) is fascinating. I think discussing this concept in the context of this manuscript is appropriate. However, the authors classify the syndrome caused by the RAF1T543M variant as a "novel segmental progeroid syndrome" in the title, abstract and first sentence of the discussion. I don't think that presented data are convincing for this classification. Indeed, two affected siblings die at very early post-natal stages (7 and 50 days, respectively) likely because of malformations that are not compatible with life and that are due to altered in uthero development. Progeroid syndromes are a heterogeneous group of syndromes, a minority of which is characterized by malformations at birth. The more general concept is that physical abnormalities are progressively acquired during post-natal life, in specific tissues that show typical features associated with aging, including senescence, decline of the stem cell compartment, increased inflammation. In the case of individuals carrying the RAF1T543M variant, none of these tissue abnormalities have been reported (due to the lack of material from patients) and the classification as a progeroid syndrome is based on external inspection of organs. Reported phenotypes are not specific and a failure of in uthero development of heart, limbs and other organs seems like the absolutely predominant trait. This in uthero phenotype is perfectly consistent with the physiological role of the MAPK pathway downstream multiple RTKs that transduce morphogenetic, in addition to mitogenic, signals. As for the authors words: "endogenous FGF/FGFR signaling is required for proper mesoderm and neural induction" and supports the author claim that the analysis of individuals carrying the RAF1T543M variant "underscores the importance of RTK signaling during human development". I think that the "progeroid" phenotypes are less clear. It is still important to present the phenotypes reminiscent of progeroid syndromes and discuss them and perhaps more clearly explain the logical connection with the increased apoptosis observed in in vitro experiments.

    In regard to the apoptosis experiment in figure 4, it supports the claim that RAF1 activity is necessary for protection from stress-induced apoptosis. However, the cartoon presented in figure 4C also shows that this process is mediated by increased ASK1/MST2 signaling. This part of the cartoon is based on the literature and has not been formally demonstrated. The authors can try to rescue the apoptotic status by silencing ASK1 in the RAF1T543M cells, similar to what has been done in Yamaguchi, O., 2004, Cardiac-specific disruption of the c-raf-1 gene induces cardiac dysfunction and apoptosis. J. Clin. Invest. 114, 937-943. Literature also suggests that RAF1-mediated inhibition of apoptosis is kinase-independent. This part is particularly interesting since the variant produces a protein that is both kinase-inactive and unstable. It would be a nice addition to the matuscript if the authors could clarify, at least in their cellular model, whether the increased apoptosis is due to the loss of either the protein or the kinase activity.

    Minor comments:

    A minor comment to the Xenopus ISH: the number of embryos that have been analyzed per condition is not reported anywhere. Figure legend explains that presented images are "representative pictures", but knowing the number of tested embryos and the penetrance of the phenotype would help undertsand the relevance of the RAF mutation in the signaling pathway under investigation.


    This work by Wong and colleagues provides conceptual advance and will be of interest for researchers dealing with RTK/MAPK signaling in multiple contexts including oncology, developmental biology and cardiomyopathy.

    The gene under investigation is widely studied in cancer, where gain-of-function, oncogenic mutations are common. The role of RAF1 during embryonic development is less known. A couple of studies have investigated the developmental phenotype of mouse models carrying either a knock-out allele (Mikula, M, et al. Embryonic lethality and fetal liver apoptosis in mice lacking the c-raf-1 gene. EMBO J. 2001. 20:1952-1962) or an allele producing a truncated protein (Wojnowski L et al., (1998) Craf-1 protein kinase is essential for mouse development. Mech Dev, 76, 141-149). A few studies have investigated the conditional inactivation in specific tissues, such as the cardiac muscle (Yamaguchi, O., 2004, Cardiac-specific disruption of the c-raf-1 gene induces cardiac dysfunction and apoptosis. J. Clin. Invest. 114, 937-943.). And one study has found heterozygous carriers of a loss-of-function mutation among cohorts of children affected by dilated cardiomyopathy (Dhandapany, P.S., et al. (2014). RAF1 mutations in childhood-onset dilated cardiomyopathy. Nat. Genet. 46, 635-639). The manuscript reports the spectrum of defects acquired during embryonic development by carriers of a pathogenic mutation in the RAF1 gene. The mutation impairs both stability and kinase activity of the protein. The manuscript points out the non-redundant role of RAF1-mediated signaling in specific organs during embryonic development.

    The person who is reviewing the manuscript has expertise in cellular signaling, mouse embryonic development and human aging.

  3. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity


    The authors describe a homozygous missense variant in RAF1 identified in a child with a lethal malformation syndrome. The T543M variant not only blocks the kinase activity, but also destabilizes the RAF1 protein. In combination, this leads not only to a loss of MAPK signaling, but also to elevated apoptosis upon cell stress, which both together very likely explain the dramatic phenotype, which may correspond to the rarely described acro-cardio-facial syndrome.

    Major comments:

    The results of the functional analysis presented by the authors are highly convincing and clearly demonstrate a LOF effect of the identified variant. The experiments are described in sufficient detail and the statistical analysis is sound. Depending on the target journal the clinical information might be a bit scarce. There are neither clinical pictures nor molecular data from the first affected sibling. For a more clinically oriented journal, a summary of clinical features in form of a table and a comparison to ACFS will be a prerequisite and facilitates appreciation of this very special phenotype. The affected child seems much older than his chronological age, which seems due to the hypoplastic viscerocranium and the loss of subcutaneous fat tissue. The heart and limb defects have no link to chronological aging. Whether such phenotypes should be really called progeroid can be debated, but it is common practice (and always a good selling point). The authors do not discuss their findings in the light of chronological aging. The hypothesis that the RAF1 LOF liberates ASK1 leading to increased apoptosis is very attractive. It would be very nice to show some experimental data proving this, but such data is not pivotal for the paper.

    Minor comments:

    The facial and overall progeroid phenotype of the affected child is quite different from most of the described ACFS patients. In contrast, the prominent neurocranium with low hairline and the hypoplastic viscerocranium remind this reviewer of Gorlin-Chaudhry-Moss/Fontaine-Petty syndrome. This differential diagnosis is also interesting since the disorder is caused by GOF variants in a mitochondrial ATP transporter increasing the sensitivity for apoptosis. This underlines the suggested link between RAF1 and the apoptosis inducer ASK1. There is probably no cellular function that has not been linked to the MAPK pathway. One of these connections is to cellular aging. Strongly activating variants in pathway members lead to oncogene-induced cellular senescence. Here, we have a progeroid phenotype, but a variant with LOF effect. This might be worthwhile to discuss. The discussion could also use some sentences on the SHSF mechanism. FGFs produced by the AER are important for proliferation of the limb mesenchyme. What is the connection between MAPK and p63?


    This is a central signaling pathway and a prominent and historically outstanding oncogene. Up to now all variants in the MAPK pathway have been described as GOF and this is the first phenotype related to a LOF. Thus, this is a landmark paper widening the view on the pathway. The paper is interesting for clinical and molecular geneticists, tumor biologists, and cell biologists. This reviewer is a biochemist and clinical geneticist with research focus on mechanisms of skeletal and connective tissue disorders.

  4. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    In the present manuscript, Wong et al describe for the first time the human phenotype resulting from the bi-allelic germline T543M mutation in the RAF1 proto-oncogene. Although somatic RAF1 mutations are outnumbered by BRAF mutations, they also occur in human cancer, but so far germline RAF1 mutations, usually dominant-acting ones abrogating RAF1 autoinhibition, have been mainly associated with RASopathies, in particular with Noonan syndrome. Here, Wong et al describe for the first time that the two individuals with a homozygous T543M mutation is associated with a neonatal lethal progeroid syndrome presenting with cutaneous, craniofacial, cardiac and limb anomalies. Moreover, the affected residue, despite its conservation across RAF1 orthologues and in the ARAF and BRAF paralogues, has neither been described as a target of somatic and germ-line mutation in cancer and RASopathies, respectively. The authors then use a combination of ectopic expression experiments in HEK293T cells and Xenopus embryos as well as CRISPR/Cas9 genome editing and structural bioinformatics to provide several lines of evidence that RAF1 T543M represents a hypomorph suppressing ERK activation. In summary, this is a very interesting and well-written manuscript with a stimulating discussion of the novel and convincing findings and the mechanisms driving the described pathology. The suggestions below might further strengthen this otherwise already very advanced manuscript.


    1. The data presented in this manuscript imply that RAF1 T543M represents a hypomorph suppressing ERK pathway activity. Nevertheless, it remains unclear whether this mutation reduces/abolishes the intrinsic activity of RAF1 or acts by a dominant-negative mechanism, e.g. by sequestrating critical components of RAF complexes, such as KSR proteins, or MEKs. To distinguish between both possibilities, it would be helpful, if the authors were able to document the intrinsic kinase activity of immunoprecipitated RAF1 T543M (and appropriate controls such as wildtype RAF1, the S257L gain-of-function allele and a truly kinase-dead variant, e.g. by mutating the aspartate of the DFG motif) towards recombinant and commercially available GST-MEK1.
    2. RAF kinases engage in complex homo- and heterodimerization events. Does RAF1 T543M form homo- and heterodimers, e.g. with BRAF or ARAF, to a similar or different extent than wildtype RAF1?


    1. Abstract. To my knowledge, RAS but not RAF was the first human oncogene identified, albeit the discovery of RAF and its viral counterparts also took place very early in the oncogene discovery era.
    2. Introduction, p. 2 "...while Braf-/- mice are embryonic lethal due to vascular defects.7" For a more balanced review, I would suggest citing additional and more recent papers describing the complex phenotypes of Braf deficient mice, e.g. PMIDs: 18332218 and PMID: 16432225
    3. Figure 1D. Here, CRAF is used instead of RAF1. As the latter is the official gene/protein name RAF1 should be used to avoid confusion to readers outside of the RAF field.


    Very interesting novel findings to researchers being active in the signalling, cancer and developmental biology fiels as well as to human geneticists and pediatricians.

    My expertise: MAPK signalling, functional characterization of oncogenic mutations