Asparagine Synthetase Deficiency causes reduced proliferation of cells under conditions of limited asparagine

doi:10.1016/j.ymgme.2015.08.007

Case Reports

. 2015 Nov;116(3):178-86.

doi: 10.1016/j.ymgme.2015.08.007. Epub 2015 Aug 14.

Asparagine Synthetase Deficiency causes reduced proliferation of cells under conditions of limited asparagine

Elizabeth Emma Palmer¹, Jaclyn Hayner², Rani Sachdev³, Michael Cardamone³, Tejaswi Kandula³, Paula Morris⁴, Kerith-Rae Dias⁴, Jiang Tao⁴, David Miller⁴, Ying Zhu⁵, Rebecca Macintosh⁶, Marcel E Dinger⁷, Mark J Cowley⁷, Michael F Buckley⁸, Tony Roscioli⁹, Ann Bye³, Michael S Kilberg², Edwin P Kirk¹⁰

Affiliations

¹ Sydney Children's Hospital, High Street Randwick NSW 2031, Australia; University of New South Wales, High Street, Sydney, NSW 2052, Australia; Genetics of Learning Disability (GOLD) service, Corner of Turton and Tinonee Roads, Waratah NSW 2298.
² Department of Biochemistry & Molecular Biology, University of Florida College of Medicine, 1200 Newell Drive, Florida, USA, 32608.
³ Sydney Children's Hospital, High Street Randwick NSW 2031, Australia; University of New South Wales, High Street, Sydney, NSW 2052, Australia.
⁴ Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia.
⁵ Genetics of Learning Disability (GOLD) service, Corner of Turton and Tinonee Roads, Waratah NSW 2298.
⁶ Sydney Children's Hospital, High Street Randwick NSW 2031, Australia.
⁷ Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia; St Vincent's Clinical School, University of New South Wales, 390 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia.
⁸ University of New South Wales, High Street, Sydney, NSW 2052, Australia; Seals Molecular Genetics, POW Hospital Campus, Barker Street, Randwick, Sydney, NSW 2031, Australia.
⁹ Sydney Children's Hospital, High Street Randwick NSW 2031, Australia; University of New South Wales, High Street, Sydney, NSW 2052, Australia; Seals Molecular Genetics, POW Hospital Campus, Barker Street, Randwick, Sydney, NSW 2031, Australia.
¹⁰ Sydney Children's Hospital, High Street Randwick NSW 2031, Australia; University of New South Wales, High Street, Sydney, NSW 2052, Australia; Seals Molecular Genetics, POW Hospital Campus, Barker Street, Randwick, Sydney, NSW 2031, Australia. Electronic address: e.kirk@unsw.edu.au.

PMID: 26318253
PMCID: PMC10152381
DOI: 10.1016/j.ymgme.2015.08.007

Case Reports

Asparagine Synthetase Deficiency causes reduced proliferation of cells under conditions of limited asparagine

Elizabeth Emma Palmer et al. Mol Genet Metab. 2015 Nov.

. 2015 Nov;116(3):178-86.

doi: 10.1016/j.ymgme.2015.08.007. Epub 2015 Aug 14.

Authors

Affiliations

¹ Sydney Children's Hospital, High Street Randwick NSW 2031, Australia; University of New South Wales, High Street, Sydney, NSW 2052, Australia; Genetics of Learning Disability (GOLD) service, Corner of Turton and Tinonee Roads, Waratah NSW 2298.
² Department of Biochemistry & Molecular Biology, University of Florida College of Medicine, 1200 Newell Drive, Florida, USA, 32608.
³ Sydney Children's Hospital, High Street Randwick NSW 2031, Australia; University of New South Wales, High Street, Sydney, NSW 2052, Australia.
⁴ Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia.
⁵ Genetics of Learning Disability (GOLD) service, Corner of Turton and Tinonee Roads, Waratah NSW 2298.
⁶ Sydney Children's Hospital, High Street Randwick NSW 2031, Australia.
⁷ Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia; St Vincent's Clinical School, University of New South Wales, 390 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia.
⁸ University of New South Wales, High Street, Sydney, NSW 2052, Australia; Seals Molecular Genetics, POW Hospital Campus, Barker Street, Randwick, Sydney, NSW 2031, Australia.
⁹ Sydney Children's Hospital, High Street Randwick NSW 2031, Australia; University of New South Wales, High Street, Sydney, NSW 2052, Australia; Seals Molecular Genetics, POW Hospital Campus, Barker Street, Randwick, Sydney, NSW 2031, Australia.
¹⁰ Sydney Children's Hospital, High Street Randwick NSW 2031, Australia; University of New South Wales, High Street, Sydney, NSW 2052, Australia; Seals Molecular Genetics, POW Hospital Campus, Barker Street, Randwick, Sydney, NSW 2031, Australia. Electronic address: e.kirk@unsw.edu.au.

PMID: 26318253
PMCID: PMC10152381
DOI: 10.1016/j.ymgme.2015.08.007

Abstract

Asparagine Synthetase Deficiency is a recently described cause of profound intellectual disability, marked progressive cerebral atrophy and variable seizure disorder. To date there has been limited functional data explaining the underlying pathophysiology. We report a new case with compound heterozygous mutations in the ASNS gene (NM_183356.3:c. [866G>C]; [1010C>T]). Both variants alter evolutionarily conserved amino acids and were predicted to be pathogenic based on in silico protein modelling that suggests disruption of the critical ATP binding site of the ASNS enzyme. In patient fibroblasts, ASNS expression as well as protein and mRNA stability are not affected by these variants. However, there is markedly reduced proliferation of patient fibroblasts when cultured in asparagine-limited growth medium, compared to parental and wild type fibroblasts. Restricting asparagine replicates the physiology within the blood-brain-barrier, with limited transfer of dietary derived asparagine, resulting in reliance of neuronal cells on intracellular asparagine synthesis by the ASNS enzyme. These functional studies offer insight into the underlying pathophysiology of the dramatic progressive cerebral atrophy associated with Asparagine Synthetase Deficiency.

Keywords: ATP binding; Asparagine; Epileptic encephalopathy; Exome sequencing; Intellectual disability.

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Figures

**Fig. 1.**
A and B: MRI scan images of the proband's brain aged 4 years. A. Sagittal T1 (A) and axial T2 (B) weighted images. Note small cranial vault with craniofacial dysmorphism in keeping with microcephaly, enlargement of the ventricles (E) and the supratentorial (A) and infratentorial (B) extra-axial spaces in keeping with global atrophy in both the supratentorial and infratentorial compartments. Diffusely thin corpus callosum (C) and slender brainstem (D). C: Head circumference of proband plotted on WHO Child Growth Standard chart (http://www.who.int/childgrowth/en/ accessed July 2015) demonstrating the head circumference was between −1 and −2 SD from the mean at birth but rapidly decelerated to be below −5 SD from the mean by the age of 20 months.

**Fig. 2.**
A: Chromosomal location of *ASNS* at 7q21.3. B: Exons of *ASNS* gene. C: Position of G337 and G289 (this patient) in the *ASNS* gene, as well as positions of pathogenic variants described in Ruzzo et al. and Ben Saleh et al. (ref. [1,2]). Variants described are spread throughout the gene in exons 3, 7, 9, 10 and 13. The variant reported by Alfadhel et al. (ref. [3] and personal communication, Alfadhel, 2015) was not in the canonical transcript but in a shorter transcript of *ASNS* (NM_001178075.1), and therefore could not be included in this diagram. D: Functional regions of the ASNS protein (adapted from Ben Saleh et al., ref. [2]). T337 and G289 (this patient) are located proximal to the ATP binding sites that make up the ATP binding pocket. E: Pedigree and chromatograms of DNA sequence changes in the *ASNS* gene. Compound heterozygous mutations were detected in the affected child (II: 1) NM_183356.3:c. [866G>C]; [1010C>T]. His father (1: 1) is a heterozygote carrier of the c.866G>C variant and his mother (II: 2) is a heterozygote carrier of the c.1010C>T variant. F: A model of human ASNS, generated with SWISS-MODEL using the glutamine-dependent bacterial ASNS-B protein as a template (8), demonstrating the location of the variants in the proband and pathogenic variants described in Ruzzo et al. and Ben Saleh et al. (ref. [1,2]). Modelling of the position of the proband's two *ASNS* variants indicates that G289 and T337 are located near the ATP binding pocket and therefore mutations at these sites could potentially alter ATP binding or hydrolysis reducing enzyme efficiency. The R550 variant described in Ruzzo et al., (1) is not included as crystallography could not be determined for the distal end of the C-terminal region and the variant described by Alfadhel et al. (3) is not included as is in a shorter, non-canonical transcript.

**Fig. 3.**
A: Regulation of the *ASNS* gene and *ASNS* mRNA stability are unaffected by the mutations. To assess *ASNS* mRNA expression and stability, fibroblasts were cultured in either complete DMEM medium (+His) or DMEM lacking histidine (−His) to activate the AAR and induced mRNA expression. Cells were collected 0, 4, 8, and 24 h post-treatment and mRNA was measured by qPCR. GAPDH mRNA, which is unaffected by histidine deprivation, was used as an internal control. Averages ± standard deviation are shown. B: ASNS protein stability in fibroblast cells is unaffected by the mutations. Fibroblasts were cultured in complete DMEM for 0, 4, 8, and 24 h. ASNS protein levels were assessed by immunoblotting. C: Cells harbouring both ASNS mutations (affected child) do not proliferate in medium depleted of asparagine. Fibroblasts were cultured in DMEM containing with 0, 0.001, 0.1, or 1 U/mL of asparaginase (ASNase). At 0, 24, and 48 h post-treatment, fibroblasts were collected by trypsin treatment and counted. Cells were grown in triplicate for each ASNase concentration and time point. Averages ± standard deviation are shown. D The stress of asparagine depletion causes an increase in ASNS expression in cells harbouring ASNS mutations. Cells were cultured in DMEM containing 0, 0.001, 0.1, and 1 U/mL ASNase. Cells were collected at 0, 4, and 8 h post-treatment and steady state ASNS mRNA was measured by qPCR. GAPDH mRNA was used as an internal control, and averages ± standard deviation are shown.

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References

1. Ruzzo EK, Capo-Chichi JM, Ben-Zeev B, Chitayat D, Mao H, Pappas AL, Hitomi Y, Lu YF, Yao X, Hamdan FF, Pelak K, Reznik-Wolf H, Bar-Joseph I, Oz-Levi D, Lev D, Lerman-Sagie T, Leshinsky-Silver E, Anikster Y, Ben-Asher E, Olender T, Colleaux L, Décarie JC, Blaser S, Banwell B, Joshi RB, He XP, Patry L, Silver RJ, Dobrzeniecka S, Islam MS, Hasnat A, Samuels ME, Aryal DK, Rodriguiz RM, Jiang YH, Wetsel WC, McNamara JO, Rouleau GA, Silver DL, Lancet D, Pras E, Mitchell GA, Michaud JL, Goldstein DB, Deficiency of asparagine synthetase causes congenital microcephaly and a progressive form of encephalopathy, Neuron 80 (2013)429–441. - PMC - PubMed
1. Ben-Salem S, Gleeson JG, Al-Shamsi AM, Islam B, Hertecant J, Ali BR, Al-Gazali L, Asparagine synthetase deficiency detected by whole exome sequencing causes congenital microcephaly, epileptic encephalopathy and psychomotor delay, Metab. Brain Dis 30 (3) (2014) 687–694. - PMC - PubMed
1. Alfadhel M, Alrifai MT, Trujillano D, Alshaalan H, Al Othaim A, Al Rasheed S, Assiri H, Alqahtani AA, Alaamery M, Rolfs A, Eyaid W, Asparagine synthetase deficiency: new inborn errors of metabolism, JIMD Rep. 22 (2015) 11–16. - PMC - PubMed
1. Richards NG, Kilberg MS, Asparagine synthetase chemotherapy, Annu. Rev. Biochem. 75 (2006) 629–654. - PMC - PubMed
1. Thorvaldsdóttir H, Robinson JT, Mesirov JP, Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration, Brief. Bioinform 14 (2013) 178–192. - PMC - PubMed

Web References

1. CADD. http://cadd.gs.washington.edu/
2. EXAC http://exac.broadinstitute.org;
3. dbSNP http://www.ncbi.nlm.nih.gov/SNP;
4. EVS http://evs.gs.washington.edu/EVS/;
5. 1000 Genomes http://www.1000genomes.org;
6. PROVEAN. http://provean.jcvi.org.

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[1] Ruzzo EK, Capo-Chichi JM, Ben-Zeev B, Chitayat D, Mao H, Pappas AL, Hitomi Y, Lu YF, Yao X, Hamdan FF, Pelak K, Reznik-Wolf H, Bar-Joseph I, Oz-Levi D, Lev D, Lerman-Sagie T, Leshinsky-Silver E, Anikster Y, Ben-Asher E, Olender T, Colleaux L, Décarie JC, Blaser S, Banwell B, Joshi RB, He XP, Patry L, Silver RJ, Dobrzeniecka S, Islam MS, Hasnat A, Samuels ME, Aryal DK, Rodriguiz RM, Jiang YH, Wetsel WC, McNamara JO, Rouleau GA, Silver DL, Lancet D, Pras E, Mitchell GA, Michaud JL, Goldstein DB, Deficiency of asparagine synthetase causes congenital microcephaly and a progressive form of encephalopathy, Neuron 80 (2013)429–441. - PMC - PubMed

[2] Ruzzo EK, Capo-Chichi JM, Ben-Zeev B, Chitayat D, Mao H, Pappas AL, Hitomi Y, Lu YF, Yao X, Hamdan FF, Pelak K, Reznik-Wolf H, Bar-Joseph I, Oz-Levi D, Lev D, Lerman-Sagie T, Leshinsky-Silver E, Anikster Y, Ben-Asher E, Olender T, Colleaux L, Décarie JC, Blaser S, Banwell B, Joshi RB, He XP, Patry L, Silver RJ, Dobrzeniecka S, Islam MS, Hasnat A, Samuels ME, Aryal DK, Rodriguiz RM, Jiang YH, Wetsel WC, McNamara JO, Rouleau GA, Silver DL, Lancet D, Pras E, Mitchell GA, Michaud JL, Goldstein DB, Deficiency of asparagine synthetase causes congenital microcephaly and a progressive form of encephalopathy, Neuron 80 (2013)429–441. - PMC - PubMed

[3] Ben-Salem S, Gleeson JG, Al-Shamsi AM, Islam B, Hertecant J, Ali BR, Al-Gazali L, Asparagine synthetase deficiency detected by whole exome sequencing causes congenital microcephaly, epileptic encephalopathy and psychomotor delay, Metab. Brain Dis 30 (3) (2014) 687–694. - PMC - PubMed

[4] Ben-Salem S, Gleeson JG, Al-Shamsi AM, Islam B, Hertecant J, Ali BR, Al-Gazali L, Asparagine synthetase deficiency detected by whole exome sequencing causes congenital microcephaly, epileptic encephalopathy and psychomotor delay, Metab. Brain Dis 30 (3) (2014) 687–694. - PMC - PubMed

[5] Alfadhel M, Alrifai MT, Trujillano D, Alshaalan H, Al Othaim A, Al Rasheed S, Assiri H, Alqahtani AA, Alaamery M, Rolfs A, Eyaid W, Asparagine synthetase deficiency: new inborn errors of metabolism, JIMD Rep. 22 (2015) 11–16. - PMC - PubMed

[6] Alfadhel M, Alrifai MT, Trujillano D, Alshaalan H, Al Othaim A, Al Rasheed S, Assiri H, Alqahtani AA, Alaamery M, Rolfs A, Eyaid W, Asparagine synthetase deficiency: new inborn errors of metabolism, JIMD Rep. 22 (2015) 11–16. - PMC - PubMed

[7] Richards NG, Kilberg MS, Asparagine synthetase chemotherapy, Annu. Rev. Biochem. 75 (2006) 629–654. - PMC - PubMed

[8] Richards NG, Kilberg MS, Asparagine synthetase chemotherapy, Annu. Rev. Biochem. 75 (2006) 629–654. - PMC - PubMed

[9] Thorvaldsdóttir H, Robinson JT, Mesirov JP, Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration, Brief. Bioinform 14 (2013) 178–192. - PMC - PubMed

[10] Thorvaldsdóttir H, Robinson JT, Mesirov JP, Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration, Brief. Bioinform 14 (2013) 178–192. - PMC - PubMed

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Asparagine Synthetase Deficiency causes reduced proliferation of cells under conditions of limited asparagine

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Asparagine Synthetase Deficiency causes reduced proliferation of cells under conditions of limited asparagine

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