Characterizing asparagine synthetase deficiency variants in lymphoblastoid cell lines

Abstract

Asparagine synthetase (ASNS) catalyzes the synthesis of asparagine (Asn) from aspartate and glutamine. Biallelic mutations in the ASNS gene result in ASNS Deficiency (ASNSD). Children with ASNSD exhibit congenital microcephaly, epileptic‐like seizures, and continued brain atrophy, often leading to premature mortality. This report describes a 4‐year‐old male with global developmental delay and seizures with two novel mutations in the ASNS gene, c.614A > C (maternal) and c.1192dupT (paternal) encoding p.H205P and p.Y398Lfs*4 variants, respectively. We employed the novel use of immortalized lymphoblastoid cell lines (LCL) to show that the proliferation of the heterozygotic parental LCL was not severely affected by culture in Asn‐free medium, but growth of the child's cells was suppressed by about 50%. Asn production by the LCL from both the father and the child was significantly decreased relative to the mother's cells. mRNA and protein analysis of the paternal LCL cells for the Y398Lfs*4 variant revealed reductions in both. Attempts to ectopically express the truncated Y398Lfs*4 variant in either HEK293T or ASNS‐null cells resulted in little or no detectable protein. Expression and purification of the H205P variant from HEK293T cells revealed enzymatic activity similar to wild‐type ASNS. Stable expression of WT ASNS rescued the growth of ASNS‐null JRS cells in Asn‐free medium and the H205P variant was only slightly less effective. However, the Y398Lfs*4 variant appeared to be unstable in JRS cells. These results indicate that co‐expression of the H205P and Y398Lfs*4 variants leads to a significant reduction in Asn synthesis and cellular growth.

Metabolomic Profiling of Asparagine Deprivation in Asparagine Synthetase Deficiency Patient-Derived Cells

“…Across fibroblast cell lines, the child had significantly lower Asn content compared to the WT, as did the father to a lesser extent, when deprived of Asn (Figure 1C). Extracellular analysis of Asn content in both LCL and fibroblasts showed significantly lower Asn in all cells deprived of Asn with the lowest extracellular Asn among the family members occurring in the child's cells (Supplementary Figures S3 and S4), which is consistent with previously reported data [22,25].…”

“…Two previous ASNSD studies utilized a lymphoblastoid immortalized cell line (LCL) model, a fibroblast model, and respective wild-type (WT) controls [22,25], which we have used for the present analysis. The LCLs are derived from a compound heterozygotic ASNSD patient with p.H205P and p.Y398Lfs*4 ASNS variants, as well as cells from the patient's mother (p.H205P) and father (p.Y398Lfs*4).…”

“…The cultured fibroblasts are derived from a different compound heterozygotic ASNSD patient with p.G373V and p.R519H ASNS variants, and the patient's mother (p.R519H) and father (p.G373V). Each of these LCL and fibroblast cell lines have been previously characterized for ASNS mRNA expression, protein stability, and activity by in cellulo and in vitro assays [22,25].…”

“…Patient-derived lymphoblastoid cell lines (LCL) were generated from an ASNSDderived compound heterozygotic child (p.H205P and p.Y398Lfs*4 ASNS variants) as well as the child's mother (p.H205P) and father (p.Y398Lfs*4), as previously reported in [25]. An unrelated wild-type EBV-immortalized human B-lymphoblastoid cell line was purchased from Cellero (1038-2750JA15, Memphis, TN, USA).…”

“…LCL and fibroblasts derived from two different families were cultured and tested as previously described [22,25]. In brief, LCL cells were maintained in RPMI 1640 supplemented with 15% FBS, P/S, and 2 mM of additional Gln.…”