In vitro functional analysis of four variants of human asparagine synthetase

Abstract

The loss‐of‐function variants of the human asparagine synthetase (ASNS) gene cause asparagine synthetase deficiency (ASNSD). Diagnosis of ASNSD requires genetic tests because a specific biochemical diagnostic for ASNSD is not available. There are a few reports describing the functional evaluation of ASNS variants. Therefore, in vitro methods are needed to evaluate the detected variants in patients. In this report, five types of human ASNS proteins (wild‐type and our reported four variants: p.Leu145Ser, p.Leu247Trp, p.Val489Asp, and p.Trp541Cysfs*5) were expressed in silkworm using a baculoviral expression system. An enzymatic activity assay of ASNS was performed, and the concentration of asparagine by ninhydrin and High Performance Liquid Chromatography methods using the purified recombinant proteins was measured. We established ASNS deficient HEK293 cells using the CRISPR/Cas9 method and evaluated the growth of cells without asparagine after transduction of ASNS variants with a lentiviral expression system. The four ASNS variants displayed significantly low enzymatic activity. The ASNS deficient HEK293 cells transduced with wild‐type ASNS grew without asparagine, whereas cells transduced with the variants did not grow or showed significantly slower growth than cells transduced with wild‐type ASNS. Herein, we established a method for evaluating the enzymatic activity of the recombinant human ASNS variants. The results of the cell‐based assay corroborated the results of the enzymatic activity. These methods should enable the evaluation of the pathogenicity of ASNS variants.

Cellular and molecular characterization of two novel asparagine synthetase gene mutations linked to asparagine synthetase deficiency

“…This result is consistent with our previous report of significantly reduced growth of fibroblasts from a heterozygotic parent expressing T337I when incubated in Asn-free medium (16). Matsumoto et al (27) investigated the effect of ASNS mutations associated with four Japanese ASNSD patients initially described by Yamamoto et al (28). For three of the variants there was a good correlation between reduced enzymatic activity and an inability to support the growth of ASNS knockout cells in Asn-free medium (27).…”

“…Matsumoto et al . ( 27 ) investigated the effect of ASNS mutations associated with four Japanese ASNSD patients initially described by Yamamoto et al . ( 28 ).…”

“…( 28 ). For three of the variants, there was a good correlation between reduced enzymatic activity and an inability to support the growth of ASNS KO cells in Asn-free medium ( 27 ). In the present report, a novel method was used to measure ASNS enzymatic activity for specific variant proteins by purification of FLAG-tagged ASNS protein from HEK293T stable cell lines.…”

“…Matsumoto et al (27) investigated the effect of ASNS mutations associated with four Japanese ASNSD patients initially described by Yamamoto et al (28). For three of the variants there was a good correlation between reduced enzymatic activity and an inability to support the growth of ASNS knockout cells in Asn-free medium (27). In the present report, a novel method was used to measure ASNS enzymatic J o u r n a l P r e -p r o o f activity for specific variant proteins by purification of FLAG-tagged ASNS protein from HEK293T stable cell lines.…”

Characterizing asparagine synthetase deficiency variants in lymphoblastoid cell lines

“…12 To date, about 75 ASNSD patients in 55 distinct families have been described. 10,[12][13][14][15][16][17][18][19][20][21] Case reports have commonly described newborns with congenital microcephaly, simplified gyral pattern, axial hypotonia, appendicular spasticity, early onset seizures, and progressive brain atrophy, often leading to premature mortality. Suspicion of ASNSD is typically prompted by recognition of the phenotype in newborns and subsequently confirmed by whole-exome DNA sequencing that identified mutations in the ASNS gene.…”