FLT4 causes developmental disorders of the cardiovascular and lymphovascular systems via pleiotropic molecular mechanisms.

AIMS: Rare, deleterious genetic variants in FLT4 are associated with Tetralogy of Fallot (TOF), the most common cyanotic congenital heart disease (CHD). Distinct genetic variants in FLT4 are also an established cause of Milroy disease, the most prevalent form of primary hereditary lymphoedema. Phenotypic features of these two conditions are non-overlapping, implying pleiotropic cellular mechanisms during development. METHODS AND RESULTS: Here, we show that FLT4 variants identified in TOF patients, when expressed in primary human endothelial cells, cause aggregation of FLT4 protein in the perinuclear endoplasmic reticulum, activating proteostatic and metabolic signalling, whereas lymphoedema-associated FLT4 variants and wildtype FLT4 do not. FLT4 TOF variants display characteristic gene expression profiles in key developmental signalling pathways, revealing a role for FLT4 in cardiogenesis distinct from its role in lymphatic development. Inhibition of proteostatic signalling abrogates these effects, identifying potential avenues for therapeutic intervention. Depletion of flt4 in zebrafish caused cardiac phenotypes of reduced heart size and altered heart looping. These phenotypes were rescued with coinjection of wildtype human FLT4 mRNA, but incompletely or not at all by mRNA harbouring FLT4 TOF variants. CONCLUSIONS: Taken together, we identify a pathogenic mechanism for FLT4 variants predisposing to TOF that is distinct from the known dominant negative mechanism of Milroy-causative variants. FLT4 variants give rise to conditions of the two circulatory subdivisions of the vascular system via distinct developmental pleiotropic molecular mechanisms. TRANSLATIONAL PERSPECTIVE: Proteostatic dysfunction, if confirmed as a mechanism of CHD pathogenesis for other predisposing genes, may identify pathways to therapeutic interventions. Distinguishing mechanistically how variants in FLT4 give rise to CHD may have potential to individualise genetic counselling in affected families.

The physiological and pathological functions of VEGFR3 in cardiac and lymphatic development and related diseases.

Vascular endothelial growth factor receptors (VEGFRs) are part of the evolutionarily conserved VEGF signalling pathways that regulate the development and maintenance of the body's cardiovascular and lymphovascular systems. VEGFR3, encoded by the FLT4 gene, has an indispensable and well-characterized function in development and establishment of the lymphatic system. Autosomal dominant VEGFR3 mutations, that prevent the receptor functioning as a homodimer, cause one of the major forms of hereditary primary lymphoedema; Milroy disease. Recently, we and others have shown that FLT4 variants, distinct to those observed in Milroy disease cases, predispose individuals to Tetralogy of Fallot, the most common cyanotic congenital heart disease, demonstrating a novel function for VEGFR3 in early cardiac development. Here, we examine the familiar and emerging roles of VEGFR3 in the development of both lymphovascular and cardiovascular systems, respectively, compare how distinct genetic variants in FLT4 lead to two disparate human conditions, and highlight the research still required to fully understand this multifaceted receptor.

Whole Exome Sequencing Reveals the Major Genetic Contributors to Nonsyndromic Tetralogy of Fallot.

RATIONALE: Familial recurrence studies provide strong evidence for a genetic component to the predisposition to sporadic, nonsyndromic Tetralogy of Fallot (TOF), the most common cyanotic congenital heart disease phenotype. Rare genetic variants have been identified as important contributors to the risk of congenital heart disease, but relatively small numbers of TOF cases have been studied to date. OBJECTIVE: We used whole exome sequencing to assess the prevalence of unique, deleterious variants in the largest cohort of nonsyndromic TOF patients reported to date. METHODS AND RESULTS: Eight hundred twenty-nine TOF patients underwent whole exome sequencing. The presence of unique, deleterious variants was determined; defined by their absence in the Genome Aggregation Database and a scaled combined annotation-dependent depletion score of ≥20. The clustering of variants in 2 genes, NOTCH1 and FLT4, surpassed thresholds for genome-wide significance (assigned as P<5×10-8) after correction for multiple comparisons. NOTCH1 was most frequently found to harbor unique, deleterious variants. Thirty-one changes were observed in 37 probands (4.5%; 95% CI, 3.2%-6.1%) and included 7 loss-of-function variants 22 missense variants and 2 in-frame indels. Sanger sequencing of the unaffected parents of 7 cases identified 5 de novo variants. Three NOTCH1 variants (p.G200R, p.C607Y, and p.N1875S) were subjected to functional evaluation, and 2 showed a reduction in Jagged1-induced NOTCH signaling. FLT4 variants were found in 2.4% (95% CI, 1.6%-3.8%) of TOF patients, with 21 patients harboring 22 unique, deleterious variants. The variants identified were distinct to those that cause the congenital lymphoedema syndrome Milroy disease. In addition to NOTCH1, FLT4 and the well-established TOF gene, TBX1, we identified potential association with variants in several other candidates, including RYR1, ZFPM1, CAMTA2, DLX6, and PCM1. CONCLUSIONS: The NOTCH1 locus is the most frequent site of genetic variants predisposing to nonsyndromic TOF, followed by FLT4. Together, variants in these genes are found in almost 7% of TOF patients.

Mitochondrial Proteins Moonlighting in the Nucleus.

Mitochondria function as cellular energy generators, producing the fuel required to drive biological processes. The response of cells to mitochondrial activity or dysfunction regulates their survival, growth, proliferation, and differentiation. Several proteins that contain mitochondrial-targeting sequences (MTS) also reside in the nucleus and there is increasing evidence that the nuclear translocation of mitochondrial proteins represents a novel pathway by which mitochondria signal their status to the cell. Here, we discuss the different mechanisms that control the dual mitochondrial and nuclear localisation of proteins and propose that these nuclear moonlighters represent a widespread regulatory circuit to maintain mitochondrial homeostasis.

A nuclear role for the respiratory enzyme CLK-1 in regulating mitochondrial stress responses and longevity.

The coordinated regulation of mitochondrial and nuclear activities is essential for cellular respiration and its disruption leads to mitochondrial dysfunction, a hallmark of ageing. Mitochondria communicate with nuclei through retrograde signalling pathways that modulate nuclear gene expression to maintain mitochondrial homeostasis. The monooxygenase CLK-1 (human homologue COQ7) was previously reported to be mitochondrial, with a role in respiration and longevity. We have uncovered a distinct nuclear form of CLK-1 that independently regulates lifespan. Nuclear CLK-1 mediates a retrograde signalling pathway that is conserved from Caenorhabditis elegans to humans and is responsive to mitochondrial reactive oxygen species, thus acting as a barometer of oxidative metabolism. We show that, through modulation of gene expression, the pathway regulates both mitochondrial reactive oxygen species metabolism and the mitochondrial unfolded protein response. Our results demonstrate that a respiratory enzyme acts in the nucleus to control mitochondrial stress responses and longevity.

A new role for an old enemy.

Drugs that change the shape of AKT, a protein kinase that promotes tumor growth, may be more effective than drugs that only target its enzymatic activity.