# Asparagine Synthetase Deficiency

> **NIH NIH R21** · UNIVERSITY OF FLORIDA · 2020 · $228,750

## Abstract

Asparagine synthetase (ASNS) catalyzes the synthesis of asparagine (Asn) from aspartate. Given ASNS
expression in most human tissues, Asn is not a dietary essential amino acid, but insufficient ASNS activity
causes tissues to become Asn-dependent. A recently identified disease, termed Asparagine Synthetase
Deficiency (ASNSD), is associated with mutations in the ASNS gene. Newborns with this disease exhibit
severe microcephaly that continues as progressive brain atrophy, intractable epileptic seizures, suppressed
development, and shortened lifespan. While exome sequencing of newborns with these symptoms has
already identified about 30-40 children, unfortunately, there is no convenient, reproducible assay for ASNS
enzymatic activity. Also, the human ASNS crystal structure has not been published, forcing the use of
molecular modeling based on the E. coli AS-B structure to predict the possible consequences of ASNSD-
associated mutations. Consequently, an “association” between ASNS mutations and disease exists, but
conclusive evidence that ASNS mutations account for the phenotype and the degree to which specific
ASNS variants function within cells remains to be established. Thus, there are critical gaps in our
knowledge of ASNS and ASNSD that are addressed as follows. Aim I hypothesis: mutations within the
human ASNS gene cause changes in protein conformation that lead to reduced enzyme activity. In
parallel, we will develop a reproducible ASNS enzymatic assay and crystallize the human ASNS wild type
protein and specific ASNSD-linked variants identified in afflicted children. Aim II hypothesis: ASNS AA
variants expressed by ASNSD children lead to suppression of cell proliferation. We will test whether
expression of specific ASNSD-associated AA variants in an ASNS-null cell support proliferation in Asn-free
medium. We will also test whether ectopic expression of wild-type ASNS protein in fibroblasts from ASNSD
children complements the disease phenotype and rescues cell growth in the absence of Asn. The
proposed studies are of high reward because the novel information gained will positively impact
the investigation, diagnosis, and treatment monitoring of ASNSD children for the following reasons.
1) Development of a direct and reproducible ASNS enzyme assay will yield much improved methods of
diagnosis and treatment monitoring for ASNSD children. 2) Structure-function studies will firmly establish
the consequences of specific ASNS mutations on protein conformation and enzyme activity. 3) The effect
of ASNSD mutations on cell growth has yet to be subjected to the expected classic characterization of an
inborn error of metabolism. For example, complementation of ASNSD-derived fibroblasts with wild type
ASNS to document rescue and, conversely, expression of ASNS AA variants in an ASNS-null cell to show
lack of growth relative to wild type enzyme. 4) Ectopic expression experiments will also reveal the impact of
specific ASNSD-associated AA variants on cell f...

## Key facts

- **NIH application ID:** 9869087
- **Project number:** 1R21HD100576-01
- **Recipient organization:** UNIVERSITY OF FLORIDA
- **Principal Investigator:** MICHAEL S. KILBERG
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $228,750
- **Award type:** 1
- **Project period:** 2020-03-01 → 2022-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9869087

## Citation

> US National Institutes of Health, RePORTER application 9869087, Asparagine Synthetase Deficiency (1R21HD100576-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9869087. Licensed CC0.

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