Functional Diversity of Human ATF6 Mutations Associated with Achromatopsia

Abstract

Purpose : Achromatopsia (ACHM) is an autosomal recessive disorder characterized by absence of cone photoreceptor function and progressive cone photoreceptor lose. We recently identified mutations in the endoplasmic reticulum (ER) stress regulator gene, ATF6, as a cause of ACHM in human patients (Kohl et al., Nature Genetics 2015). In response to ER stress, ATF6 undergoes regulated intramembrane proteolysis to release its cytosolic bZIP transcriptional activator domain to transcriptionally upregulate genes encoding chaperones and enzymes required for ER protein folding and ER homeostasis, such as GRP78/BiP. Here, we functionally characterized the molecular defects of all known ACHM-associated ATF6 mutations.

Methods : We performed functional studies assessing various ATF6 mutations, including missense mutations, splice variants, and truncated ATF6 mutants. Recombinant ATF6 genes carrying different mutations were created by site-direct mutagenesis and expressed in HEK293 cells. Primary fibroblasts were collected from two ACHM families with different ATF6 mutations (p.R324C and p.Y567N). ATF6 mutants were assessed for their abilities to traffic from ER to Golgi, undergo proteolytic cleavage, and transcriptionally upregulate downstream target genes (i.e. GRP78/BiP).

Results : All the ATF6 mutations are classified into three functional classes. Class I ATF6 mutants have absent or defective bZip domains, causing loss of transcriptional activity. Class II ATF6 mutants produce truncated cytosolic ATF6 fragments with intact transcriptional activator/bZIP domains of ATF6 that constitutively upregulate downstream target genes even in the absence of ER stress. Class III ATF6 mutants show impaired ER to Golgi trafficking and therefore undergo diminished regulated intramembrane proteolysis in response to ER stress. Furthermore, class I and class II mutant ATF6 mRNAs with premature stop codons may undergo nonsense mediated decay.

Conclusions : The functional diversity of ATF6 mutants suggests that multiple mechanisms can contribute to the pathology of ACHM caused by mutations in ATF6 gene.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.