The Role of Methionine Aminopeptidase 2 in Lymphangiogenesis.

During the metastasis process, tumor cells invade the blood circulatory system directly from venous capillaries or indirectly via lymphatic vessels. Understanding the relative contribution of each pathway and identifying the molecular targets that affect both processes is critical for reducing cancer spread. Methionine aminopeptidase 2 (MetAp2) is an intracellular enzyme known to modulate angiogenesis. In this study, we investigated the additional role of MetAp2 in lymphangiogenesis. A histological staining of tumors from human breast-cancer donors was performed in order to detect the level and the localization of MetAp2 and lymphatic capillaries. The basal enzymatic level and activity in vascular and lymphatic endothelial cells were compared, followed by loss of function studies determining the role of MetAp2 in lymphangiogenesis in vitro and in vivo. The results from the histological analyses of the tumor tissues revealed a high MetAp2 expression, with detectable sites of co-localization with lymphatic capillaries. We showed slightly reduced levels of the MetAp2 enzyme and MetAp2 mRNA expression and activity in primary lymphatic cells when compared to the vascular endothelial cells. The genetic and biochemical manipulation of MetAp2 confirmed the dual activity of the enzyme in both vascular and lymphatic remodulation in cell function assays and in a zebrafish model. We found that cancer-related lymphangiogenesis is inhibited in murine models following MetAp2 inhibition treatment. Taken together, our study provides an indication that MetAp2 is a significant contributor to lymphangiogenesis and carries a dual role in both vascular and lymphatic capillary formation. Our data suggests that MetAp2 inhibitors can be effectively used as anti-metastatic broad-spectrum drugs.

Bi-allelic Truncating Mutations in CEP78, Encoding Centrosomal Protein 78, Cause Cone-Rod Degeneration with Sensorineural Hearing Loss.

Inherited retinal diseases (IRDs) are a diverse group of genetically and clinically heterogeneous retinal abnormalities. The present study was designed to identify genetic defects in individuals with an uncommon combination of autosomal recessive progressive cone-rod degeneration accompanied by sensorineural hearing loss (arCRD-SNHL). Homozygosity mapping followed by whole-exome sequencing (WES) and founder mutation screening revealed two truncating rare variants (c.893-1G>A and c.534delT) in CEP78, which encodes centrosomal protein 78, in six individuals of Jewish ancestry with CRD and SNHL. RT-PCR analysis of CEP78 in blood leukocytes of affected individuals revealed that the c.893-1G>A mutation causes exon 7 skipping leading to deletion of 65bp, predicted to result in a frameshift and therefore a truncated protein (p.Asp298Valfs(∗)17). RT-PCR analysis of 17 human tissues demonstrated ubiquitous expression of different CEP78 transcripts. RNA-seq analysis revealed three transcripts in the human retina and relatively higher expression in S-cone-like photoreceptors of Nrl-knockout retina compared to rods. Immunohistochemistry studies in the human retina showed intense labeling of cone inner segments compared to rods. CEP78 was reported previously to interact with c-nap1, encoded by CEP250 that we reported earlier to cause atypical Usher syndrome. We conclude that truncating mutations in CEP78 result in a phenotype involving both the visual and auditory systems but different from typical Usher syndrome.

Genotype-functional-phenotype correlations in photoreceptor guanylate cyclase (GC-E) encoded by GUCY2D.

The GUCY2D gene encodes for the photoreceptor guanylate cyclase GC-E that synthesizes the intracellular messenger of photoreceptor excitation cGMP and is regulated by intracellular Ca2+-sensor proteins named guanylate cyclase-activating proteins (GCAPs). Over 140 disease-causing mutations have been described so far in GUCY2D, 88% of which cause autosomal recessive Leber congenital amaurosis (LCA) while heterozygous missense mutations cause autosomal dominant cone-rod degeneration (adCRD). Mutations in GUCY2D are one of the major causes of all LCA cases and are the major cause of adCRD. A single amino acid, arginine at position 838, is likely to be the most sensitive one in GC-E as four single mutations and two complex mutations were reported to affect R838. The biochemical effect of 45 GC-E variants was studied showing a clear genotype-phenotype correlation: LCA-causing mutations either show reduced ability or complete inability to synthesize cGMP from GTP, while CRD-causing mutations are functional, but shift the Ca2+-sensitivity of the GC-E - GCAP complex. Eight animal models of retinal guanylate cyclase deficiency have been reported including knockout (KO) mouse and chicken models. These two models were used for gene augmentation therapy that yielded promising results. Here we integrate the available information on the genetics, biochemistry and phenotype that is related to GUCY2D mutations. These data clearly show that mutation type (missense versus null) and localization (dimerization domain versus other protein domains) are correlated with the pattern of inheritance, impact on enzymatic function and retinal phenotype. Such clear correlation is unique to GUCY2D while mutations in many other retinal disease genes show variable phenotypes and lack of available biochemical assays.