Endoglin mutants retained in the endoplasmic reticulum exacerbate loss of function in hereditary hemorrhagic telangiectasia type 1 (HHT1) by exerting dominant negative effects on the wild type allele.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder affecting 1 in 5000-8000 individuals. Hereditary hemorrhagic telangiectasia type 1 (HHT1) is the most common HHT and manifests as diverse vascular malformations ranging from mild symptoms such as epistaxis and mucosal and cutaneous telangiectases to severe arteriovenous malformations (AVMs) in the lungs, brain or liver. HHT1 is caused by heterozygous mutations in the ENG gene, which encodes endoglin, the TGFß homodimeric co-receptor. It was previously shown that some endoglin HHT1-causing variants failed to traffic to the plasma membrane due to their retention in the endoplasmic reticulum (ER) and consequent degradation by ER-associated degradation (ERAD). Endoglin is a homodimer formed in the ER, and we therefore hypothesized that mixed heterodimers might form between ER-retained variants and WT protein, thus hampering its maturation and trafficking to the plasma membrane causing dominant negative effects. Indeed, HA-tagged ER-retained mutants formed heterodimers with Myc-tagged WT endoglin. Moreover, variants L32R, V105D, P165L, I271N and C363Y adversely affected the trafficking of WT endoglin by reducing its maturation and plasma membrane localization. These results strongly suggest dominant negative effects exerted by these ER-retained variants aggravating endoglin loss of function in patients expressing them in the heterozygous state with the WT allele. Moreover, this study may help explain some of the variability observed among HHT1 patients due to the additional loss of function exerted by the dominant negative effects in addition to that due to haploinsufficiency. These findings might also have implications for some of the many conditions impacted by ERAD.

Endoglin Wild Type and Variants Associated With Hereditary Hemorrhagic Telangiectasia Type 1 Undergo Distinct Cellular Degradation Pathways.

Endoglin, also known as cluster of differentiation 105 (CD105), is an auxiliary receptor in the TGFß signaling pathway. It is predominantly expressed in endothelial cells as a component of the heterotetrameric receptor dimers comprising type I, type II receptors and the binding ligands. Mutations in the gene encoding Endoglin (ENG) have been associated with hereditary hemorrhagic telangiectasia type 1 (HHT1), an autosomal dominant inherited disease that is generally characterized by vascular malformation. Secretory and many endomembrane proteins synthesized in the Endoplasmic reticulum (ER) are subjected to stringent quality control mechanisms to ensure that only properly folded and assembled proteins are trafficked forward through the secretory pathway to their sites of action. We have previously demonstrated that some Endoglin variants causing HHT1 are trapped in the ER and fail to traffic to their normal localization in plasma membrane, which suggested the possible involvement of ER associated protein degradation (ERAD) in their molecular pathology. In this study, we have investigated, for the first time, the degradation routes of Endoglin wild type and two mutant variants, P165L and V105D, and previously shown to be retained in the ER. Stably transfected HEK293 cells were treated with proteasomal and lysosomal inhibitors in order to elucidate the exact molecular mechanisms underlying the loss of function phenotype associated with these variants. Our results have shown that wild type Endoglin has a relatively short half-life of less than 2 hours and degrades through both the lysosomal and proteasomal pathways, whereas the two mutant disease-causing variants show high stability and predominantly degrades through the proteasomal pathway. Furthermore, we have demonstrated that Endoglin variants P165L and V105D are significantly accumulated in HEK293 cells deficient in HRD1 E3 ubiquitin ligase; a major ERAD component. These results implicate the ERAD mechanism in the pathology of HHT1 caused by the two variants. It is expected that these results will pave the way for more in-depth research studies that could provide new windows for future therapeutic interventions.

Endoplasmic Reticulum Associated Protein Degradation (ERAD) in the Pathology of Diseases Related to TGFß Signaling Pathway: Future Therapeutic Perspectives.

The transforming growth factor signaling pathway (TGFß) controls a wide range of cellular activities in adulthood as well as during embryogenesis including cell growth, differentiation, apoptosis, immunological responses and other cellular functions. Therefore, germline mutations in components of the pathway have given rise to a heterogeneous spectrum of hereditary diseases with variable phenotypes associated with malformations in the cardiovascular, muscular and skeletal systems. Our extensive literature and database searches revealed 47 monogenic diseases associated with germline mutations in 24 out of 41 gene variant encoding for TGFß components. Most of the TGFß components are membrane or secretory proteins and they are therefore expected to pass through the endoplasmic reticulum (ER), where fidelity of proteins folding is stringently monitored via the ER quality control machineries. Elucidation of the molecular mechanisms of mutant proteins' folding and trafficking showed the implication of ER associated protein degradation (ERAD) in the pathogenesis of some of the diseases. For example, hereditary hemorrhagic telangiectasia types 1 and 2 (HHT1 and HHT2) and familial pulmonary arterial hypertension (FPAH) associated with mutations in Endoglin, ALK1 and BMPR2 components of the signaling pathway, respectively, have all exhibited loss of function phenotype as a result of ER retention of some of their disease-causing variants. In some cases, this has led to premature protein degradation through the proteasomal pathway. We anticipate that ERAD will be involved in the mechanisms of other TGFß signaling components and therefore warrants further research. In this review, we highlight advances in ER quality control mechanisms and their modulation as a potential therapeutic target in general with particular focus on prospect of their implementation in the treatment of monogenic diseases associated with TGFß components including HHT1, HHT2, and PAH. In particular, we emphasis the need to establish disease mechanisms and to implement such novel approaches in modulating the molecular pathway of mutant TGFß components in the quest for restoring protein folding and trafficking as a therapeutic approach.

A novel aberrant splice site mutation in COL27A1 is responsible for Steel syndrome and extension of the phenotype to include hearing loss.

Steel syndrome is an autosomal recessive disease characterized by skeletal abnormalities and dysmorphic features. The first mutation associated with this syndrome was reported in Puerto Rican children. In this study, we identified a novel homozygous splice site variant in COL27A1 (c.3556-2A>G) in a consanguineous Emirati family with a child affected by Steel syndrome. In addition, the affected child had severe non-progressive sensorineural hearing loss not reported previously. The variant segregated in the family in an autosomal recessive manner and we show that the variant alters mRNA splicing. Furthermore, relative quantitative analysis revealed a marked reduction in gene expression in the proposita compared to healthy controls. Segregation analysis of heterozygous variants, related to hearing loss, identified by whole exome sequencing in the child (ILDR1: c.1159T>C, SYNE4: c.313G>C, and GPR98: c.18746T>G) excluded them from being responsible for the hearing loss in the proposita. In addition, the products of these genes are not interacting in the same pathway and have only been reported to cause deafness in an autosomal recessive manner. Therefore, we conclude that the novel splice-site variant identified in COL27A1 is the most likely cause for Steel syndrome in this family and that the hearing loss is part of this syndrome's phenotype.