ADrosophilanatural variation screen identifies NKCC1 as a substrate of NGLY1 deglycosylation and a modifier of NGLY1 deficiency

Abstract

15N-Glycanase 1 (NGLY1) is a cytoplasmic deglycosylating enzyme. Loss-of-function mutations in 16the NGLY1 gene cause NGLY1 deficiency, which is characterized by developmental delay, 17seizures, and a lack of sweat and tears. To model the phenotypic variability observed among 18 patients, we crossed a Drosophila model of NGLY1 deficiency onto a panel of genetically diverse 19 strains. The resulting progeny showed a phenotypic spectrum from 0-100% lethality. Association 20analysis on the lethality phenotype as well as an evolutionary rate covariation analysis generated 21lists of modifying genes, providing insight into NGLY1 function and disease. The top association 22hit was Ncc69 (human NKCC1/2), a conserved ion transporter. Analyses in NGLY1 -/-mouse 23 cells demonstrated that NKCC1 is misglycosylated and has reduced function, making it only the 24 second confirmed NGLY1 enzymatic substrate. The misregulation of this ion transporter may 25 explain the observed defects in secretory epithelium function in NGLY1 deficiency patients. 26

Natural genetic variation inDrosophila melanogasterreveals genes associated withCoxiella burnetiiinfection

“…These analyses were not sufficiently powered to detect polymorphisms at a Bonferroni-corrected P -value of 2.64 × 10 −8 . Therefore, we employed a genome-wide suggestive P -value threshold of 10 −5 which has been used for studies employing the DGRP ( He et al 2014 ; Chow et al 2016 ; Kelsey and Clark 2017 ; Schmidt et al 2017 ; Lavoy et al 2018 ; Mackay and Huang 2018 ; Palu et al 2020 ; Talsness et al 2020 ). Using this P -value, we obtained a total of 69 associated polymorphisms from the GWA analyses, which included five duplicate variants ( Figure 2 and Supplementary Table S5 ).…”

Evolutionary rate covariation is pervasive between glycosylation pathways and points to potential disease modifiers

“…CDG patients have a broad clinical spectrum influenced by different disease-causing variants, environmental factors, and genetic background (63,64). Previous attempts to identify genetic modifiers with standard methods (i.e., mutagenesis screens and using natural variation in the population) have found promising candidates (14)(15)(16)20). These time and effort-intensive methods can only be applied to one disorder at a time.…”

“…GPAA1, a 368 member of the GPI transamidase complex (13), had 369 high ERC with ALG1, a mannosyltransferase in N-370 linked glycosylation (ERC = 9.29) (11). All seven 371 glycosylation proteins in these top-scoring pairs are 372 associated with their own CDGs, further suggesting 373 that known CDG genes are likely candidates for 374 genetic modifiers of other CDGs (14)(15)(16)20). 375 Top scores in our dataset revealed high ERC between PIGB and UTP20 (ERC = 10.33) and PIGN and WDR36 (ERC = 9.74).…”

“…In 2002, a study examined a small group of PMM2-CDG patients, the most common CDG, and identified a variant in ALG6 , an N-linked glycosylation gene, segregating with more severe outcomes (20). A modifier screen and ERC analysis of NGLY1, a deglycosylating enzyme associated with a CDG, identified multiple glycosylation genes showing high ERC with NGLY1, implicating them as potential modifiers (15). A CRISPR screen in cells with reduced DPAGT1 function, the first step in N-glycan synthesis, identified multiple glycosylation genes able to rescue glycosylation defects caused by inhibition of DPAGT1 (14).…”

“…Standard methods for identifying genetic modifiers typically involve utilizing natural variation in the population or performing large mutagenesis screens (1). Several recent studies reported modifier screens in animal models of specific CDGs (14)(15)(16). However, with over 150 CDGs, performing similar studies for each CDG is impractical.…”