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PIGW-CDG Cure Odyssey
When pigs fly, the saying goes. There's a group of congenital disorders of glycosylation known as the PIG's, and they've taken flight with yeast-powered drug repurposing screens, including PIGW-CDG.
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PIGW-CDG is one of the rarest CDGs. It is caused by homozygous loss-of-function mutations in the PIGW gene. PIG is short for phosphatidylinositol glycan anchor biosynthesis. The PIG genes literally form an alphabet soup — with perhaps the exception of PIGZ. Perlara has already worked on or is currently working on PIGA-CDG, PIGS-CDG, PIGN-CDG and PGAP3-CDG. At this point, no other biotech company on the planet is working on as many PIG’s, let alone CDGs. In some ways, we’re just getting started.
Fewer than ten cases of PIGW-CDG are described in the medical literature. Hannes from the US is one of them. Dr Eva Morava-Kozicz — affectionately, the Queen of CDG — introduced us to Hannes’s mom Kelsey at the end of last year. After several rounds of discussion, a yeast-powered drug repurposing project was launched at the start of this year.
As shown in the excerpted figure above, the PIGW gene encodes an inositol acyltransferase, which adds a temporary third acyl chain to the inositol ring at the base of the growing GPI anchor. PIGW deficiency is primarily characterized by intellectual disability, epileptic seizures and hypotonia. Hannes’ PIGW gene is compound heterozygous for two missense mutations, R36G and P108L, both of which are technically classified as VUS’s, or Variants of Uncertain Significance. The yeast version of PIGW is called GWT1 (GPI-anchored Wall protein Transfer).
The P108L mutation replaces an evolutionarily conserved proline (P) with a leucine (L) residue which may compromise protein function. This variant is not present in population databases (e.g., gnomAD) and has not been reported in the literature in individuals affected by PIGW-related conditions. The R36G mutation has been reported in several affected individuals. The R36 position is not evolutionarily conserved between humans and yeast, however both arginine (R; human) and tryptophan (W; yeast) substitution with glycine (R36G) is predicted to be damaging.
Both of Hannes’ mutations are computationally predicted to be damaging but the exact defect or defects caused by each mutation – protein misfolding, protein instability, protein mis-localization, catalytic site inactivation, among others – have not yet been elucidated. Here’s the 3-D structure of PIGW as predicted by AlphaFold.
Because PIGW-CDG is so under-studied, many open questions remain. Why does nature have a third acyl chain transiently attached to inositol during GPI anchor synthesis? Does the maturation of all of the 150+ GPI anchor proteins in the human proteome require this transient third acyl chain? Or are just some GPI anchor proteins vulnerable?
Unlike other GPI anchor CDGs, PIGW doesn’t always present with elevated alkaline phosphatase (ALP) as reported in this 2016 study by Dr Thorsten Marquadt and colleagues. An earlier study from 2003 showed that several representative GPI anchor proteins were reduced in surface expression in a generic cell line where PIGW was knocked out. Other than that, there’s not much else out there.
As with the other CDG projects we launched in 2022, our intention was to generate Hannes yeast avatars. With the exception of PMM2-CDG where we had a ton of previous experience working with the gene in question, all of our other attempts at novel yeast avatar generation ran into the buzzsaw of unexpected troubleshooting challenges. When we have to pivot, our job is to explain to our partner pioneer families why, answer all of their questions and address all of their concerns. Two months ago, Perlara Cure Guides Dr. Kristin Kantautas and Dr. Kate Rosenbalm explained to Kelsey why we decided to pivot to a temperature-sensitive yeast avatar.
Thankfully, a temperature-sensitive GWT1 mutant — known as gwt1-20 — was available from the Euroscarf collection. When it arrived in the lab, it displayed a growth defect at 35˚C, as expected. When we sequenced its GWT1 gene, the expected mutations turned up in the correct locations. While there isn’t a human living with PIGW-CDG who carries these exact mutations, we were confident from other project pivots to temperature-sensitive yeast avatars that having a yeast mutant in which PIGW function can be reduced instantly by temperature shift was more important than the exact identity of the variants.
Round one of Z’ optimization didn’t quite go as anticipated because the wildtype strain exhibited less growth than usual, and the PIGW temperature-sensitive yeast avatar more growth than desired. The remedy was to thaw a fresh stock of the wildtype positive control. It’s the yeast experimental equivalent of pressing the reset button.
The second go-around proved successful, with excellent separation between wildtype and the PIGW temperature-sensitive yeast avatar. In order to be totally sure, we repeated the Z’ optimization experiment a third time. This is why we now explicitly include the cost of at least one additional round of Z’ optimization experiments in our Statements of Work. We plan for each experiment to work the first time, but we budget for unexpected troubleshooting.
Onward to the TargetMol screen, which is scheduled to take place next week at HTSF if all goes according to schedule.
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