New @chowlab paper about #NGLY1 up on @biorxivpreprint! A big effort between postdoc @dana_talsness and grad student @kgowings. With important help from Aylin Rodan & @NathanClark111’s groups at @UofUMedicine & Cat Lutz’s group at @jacksonlab. https://www.biorxiv.org/content/10.1101/2020.04.13.039651v1

A few years ago we set out to find modifiers of #NGLY1 deficiency in Drosophila. We performed a natural genetic variation screen and found that lethality associated with loss of NGLY1 deficiency can range from 0-100% lethality dependent of genetic background. WOW!

We used this quantitative variation to run a genome wide association and identified a number of potential modifiers. We found a number of genes with proteostasis functions, including 3 genes with known interactions with NGLY1. Awesome proof of principle.

We also ran a GSEA to try to find hidden enrichment from the GWAS results. We identified a number of enriched categories like nuclear pore complex and ncRNA, but these made little sense.

We decided to ask if evolution could help contextualize these GSEA results. so we collaborated with @NathanClark111 and use his Evolutionary Rate Covariation method to identify NGLY1 co-evolving genes. Thinking that co-evolving genes might overlap with genes from GWAS and GSEA

To our surprise, when we looked at what functions were enriched among genes co-evolving with NGLY1, we found enrichment in (you guessed it!) nuclear pore complex and ncRNA!!!! WOW! the same genes that co-elve with NGLY1 also contribute to variable phenotypes.

We also found an enrichment for Congenital Disorders of Glycosylation (CDG) genes among genes coevolving with NGLY1. While NGLY1 is a CDG gene, there is no reason to expect that this clinical grouping of genes would co-evolve, they are spread across pathways and parts of the cell

For this study, we decided to follow up on one candidate from the GWAS, Ncc69 - the top candidate with a human ortholog - NKCC1/2. This gene is a Na/K/Cl co-transporter. This was confusing. How would this channel interact with a glycosylation gene?

We were so LUCKY that one of the world's experts in Ncc69 and NKCC1/2, Aylin Rodan, had her lab on the 2nd floor of our building! OMG! Thanks to their expertise, we were able to show a number of genetic interactions between NGLY1 and Ncc69 in the fly, validating our GWAS result.

We next wanted to know if this interaction was conserved in mouse. Fortunately, Cat Lutz's group at @jacksonlab had made the NGLY1 -/- mice and sent us MEFs.

We probed for NKCC1 in the MEFs and found that the channel consistently ran at a higher MW than in +/+ cells! WOW! the size difference is consistent with a single N-glycosylation event. treatment with different glycosylation enzymes the N-glycosylation difference.

We reasoned that if there is a glycosylation difference, there must be a functional difference. Again, the Rodan lab helped us show that there is a 50% reduction in NKCC1 activity in the -/- MEFs!

There is a conserved interaction between NGLY1/NKCC1 in flies and mice!

The striking thing about this finding is that NKCC1 has a syndrome associated with it that has many overlapping features with NGLY1, including lack of tear, sweat and saliva production and abnormal ABRs.

We think this interaction helps explain some of these NGLY1 symptoms

because NKCC1 is a channel, we think it's a good druggable target and we plan on looking for small molecules that might increase activity.

One of the reasons I really love this story is because it so clearly demonstrates the value of forward genetics in Drosophila to identify promising conserved disease elements. this is esp important for rare disease where time is of the essence.