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

Staklinski, Stephen J.; Chang, Mario C.; Yu, Fang; Ruff, Kathleen Collins; Franz, David Neal; Qian, Zhijian; Bloom, Linda B.; Merritt, Matthew E.; McKenna, Robert; Kilberg, Michael S.

doi:10.1016/j.jbc.2022.102385

Cited by 9 publications

(29 citation statements)

References 37 publications

(85 reference statements)

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“…Total cellular RNA was isolated and analyzed for specific mRNA species by quantitative real-time PCR (qRT-PCR) as described. 24 Primers used for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were: forward 5 0 -TTGGTATCGTGGAAGGACTC-3 0 and reverse 5 0 -ACAGTCTTCTGGGTGGCAGT-3 0 . Primers used for ASNS mRNA were: forward 5 0 -GCAGCTGAAAGAAGCCCAAGT-3 0 and reverse 5 0 -TGTCTTCCATGCCAATTGCA-3 0 .…”

Section: Rna Isolation and Quantitative Real-time Pcrmentioning

confidence: 99%

“…Protein analysis by immunoblotting was performed as previously described. 24 Antibody dilutions were as follows: 1:200 ASNS mouse monoclonal antibody, 30 1:1000 FLAG primary antibody (Cell Signaling no. 147935), 1:5000 antimouse IgG HRP-conjugated secondary antibody (Bio-Rad no.…”

Section: Immunoblottingmentioning

confidence: 99%

“…Cell pellets and media samples were processed and analyzed by gas chromatography-mass spectrometry (GC-MS), as reported previously. 24,31,32 GC-MS data acquisition was accomplished with a Thermo Scientific Single Quadrupole Mass Spectrometer (ISQ) and Gas Chromatograph (Trace 1310). Amino acid peak areas were processed with XCalibur Quan Browser software and normalized to the peak area of a DL-norleucine internal standard.…”

Section: Gas Chromatography-mass Spectrometry Analysis Of Asnmentioning

confidence: 99%

“…F2426) as described. 24 The purity of each protein preparation was confirmed by SDS-PAGE and Coomassie Brilliant Blue R-250 staining (Bio-Rad 1 no. 61-0400) and aliquots were stored at À80 C until thawed only once for enzyme activity analysis.…”

Section: Purification Of Asns Proteins From Flag-asns Expressing Hek2...mentioning

confidence: 99%

“…These studies document the biochemical and cellular consequences using protein modeling, purification of variant proteins, and growth analysis using immortalized, cultured cells from the child and parents. Although our previous ASNSD reports involved patient primary fibroblasts in culture, [22][23][24] the present investigation utilizes the novel approach of immortalized B-lymphocytes to provide ASNSD lymphoblastoid cell lines (LCL) as a cell culture model. To establish the physiological consequences of each variant in a genetically uniform background and independent of expression of wild-type (WT) protein or another variant, each variant protein was also ectopically expressed and characterized in an ASNS-null cell line.…”

Section: Introductionmentioning

confidence: 99%

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Characterizing asparagine synthetase deficiency variants in lymphoblastoid cell lines

et al. 2023

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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.

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Section: Rna Isolation and Quantitative Real-time Pcrmentioning

confidence: 99%

Section: Immunoblottingmentioning

confidence: 99%

Section: Gas Chromatography-mass Spectrometry Analysis Of Asnmentioning

confidence: 99%

Section: Purification Of Asns Proteins From Flag-asns Expressing Hek2...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Characterizing asparagine synthetase deficiency variants in lymphoblastoid cell lines

et al. 2023

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A novel variant in ASNS gene responsible for syndromic intellectual disability and microcephaly: Case report and literature review

Jahanpanah,

Mokhtari,

Mokaber

et al. 2024

Molec Gen & Gen Med

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BackgroundThe ASNS (ASNS, MIM 108370) gene variations are responsible for asparagine synthetase deficiency (ASNSD, MIM 615574), a very rare autosomal recessive disease characterized by cerebral anomalies. These patients have congenital microcephaly, progressive encephalopathy, severe intellectual disability, and intractable seizures.MethodClinical characteristics of the patient were collected. Exome sequencing was used for the identification of variants. Sanger sequencing was used to confirm the variant in the target region. The structure of the protein was checked using the DynaMut2 web server.ResultsThe proband is an 11‐year‐old Iranian‐Azeri girl with primary microcephaly and severe intellectual disability in a family with a consanguineous marriage. Symptoms emerged around the 10–20th days of life, when refractory epileptic gaze and unilateral tonic–clonic seizures initiated without any provoking factor such as fever. A brain MRI revealed no abnormalities except for brain atrophy. The karyotype was normal. Using exome sequencing, we identified a novel homozygous variant of thymine to adenine (NM_001673.5:c.538T>A) in the ASNS gene. Both parents had a heterozygous variant in this location. Subsequently, Sanger sequencing confirmed this variant. We also reviewed the clinical manifestations and MRI findings of the previously reported patients.ConclusionIn the present study, a novel homozygous variant was recognized in the ASNS gene in an Iranian‐Azeri girl manifesting typical ASNSD symptoms, particularly intellectual disability and microcephaly. This study expands the mutation spectrum of ASNSD and reviews previously reported patients.

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A method for measurement of human asparagine synthetase (ASNS) activity and application to ASNS protein variants associated with ASNS deficiency

Chang,

Staklinski,

Merritt

et al. 2023

Biology Methods and Protocols

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Human asparagine synthetase (ASNS) catalyzes the conversion of aspartate to asparagine in an ATP-dependent reaction that utilizes glutamine as a nitrogen source while generating glutamate, AMP, and pyrophosphate as additional products. Asparagine Synthetase Deficiency (ASNSD) is an inborn error of metabolism in which children present with homozygous or compound heterozygous mutations in the ASNS gene. These mutations result in ASNS variant protein expression. It is believed that these variant ASNS proteins have reduced enzymatic activity or stability resulting in a lack of sufficient asparagine production for cell function. Reduced asparagine production by ASNS appears to severely hinder fetal brain development. Although a variety of approaches for assaying ASNS activity have been reported, we present here a straightforward method for the in vitro enzymatic analysis by detection of AMP production. Our method overcomes limitations in technical feasibility, signal detection, and reproducibility experienced by prior methods like high-performance liquid chromatography (HPLC), ninhydrin staining, and radioactive tracing. After purification of FLAG-tagged R49Q, G289A, and T337I ASNS variants from stably expressing HEK 293 T cells, this method revealed a reduction in activity of 90%, 36%, and 96% respectively. Thus, ASNS protein expression and purification, followed by enzymatic activity analysis, has provided a relatively simple protocol to evaluate structure-function relationships for ASNS variants reported for ASNSD patients.

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

Cited by 9 publications

References 37 publications

Characterizing asparagine synthetase deficiency variants in lymphoblastoid cell lines

Characterizing asparagine synthetase deficiency variants in lymphoblastoid cell lines

A novel variant in ASNS gene responsible for syndromic intellectual disability and microcephaly: Case report and literature review

A method for measurement of human asparagine synthetase (ASNS) activity and application to ASNS protein variants associated with ASNS deficiency

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