Secondary Logo

Journal Logo

RESEARCH ARTICLES

CRISPR/Cas9-mediated mutation of asparagine-linked glycosylation 13 transcript variant 1 causes epilepsy in mice

Yu, Baolia,b; Zhu, Gengc,d; Li, Shangange; Chen, Xuejine; Fang, Qiana; Zhang, Yanf; Liu, Yixung; Li, Shengtianc,*; Wu, Jia,b,*

Author Information
Journal of Bio-X Research: March 2020 - Volume 3 - Issue 1 - p 6-12
doi: 10.1097/JBR.0000000000000059

Abstract

Introduction

The asparagine-linked glycosylation 13 homolog (Alg13), which is located on the X-chromosome, encodes the Alg13 protein, which contains a predicted catalytic domain but lacks a membrane-spanning domain.[1] Alg13 is recruited by Alg14 to form a bipartite uridine 5′-diphospho-glucuronosyltransferase (UDP)-N-acetylglucosamine (GlcNAc) transferase on the cytoplasmic surface of the endoplasmic reticulum (ER), where it catalyzes the formation of GlcNAc2-PP-dol.[2] The UDP-GlcNAc transferase contains at least two different polypeptides that have catalytic and membrane anchoring functions.[3] Overexpression of Alg13 results in cytoplasmic partitioning and down-regulation of Alg14 expression. However, up-regulation of Alg14 inhibits the anchoring function of transferase, indicating that Alg14 plays a critical role in regulating accurate localization of Alg13 to the ER.[4]

Alg13 plays an important role in the multistep N-glycosylation pathway, which enables dolichol-glycan assembly and glycan transfer to nascent proteins in the ER, and functions in an identical manner for all N-glycosylated proteins.[5] Defects in any of the proteins involved in the glycosylation machinery normally lead to loss or shortening of oligosaccharide moieties linked to glycoproteins, and are termed congenital disorders of glycosylation type I (CDG-I). Abnormalities in glycosylation-related proteins lead to loss or shortening of other oligosaccharide molecules associated with glycosylation, thus inducing CDG-I. Patients with CDG-I often present with epilepsy, mental retardation, coagulopathy, cerebellar hypoplasia and dysmorphia.[6–10]

Although CDG-I has been known of for decades, little is known about the pathophysiology of this group of disorders. At present, diagnosing patients with CDG-I mainly relies on analysis of serum, fibroblasts and leukocytes, as the pathological causes of the disease are still poorly understand. Animal models could help address this problem. Several CDG-I mouse models, such as CDG-Ia (containing a mutation in PMM2),[6] CDG-Ib (containing a mutation in Mpi),[7] CDG-Iq (containing a mutation in SRD5A3)[8,9] and CDG-Ij (containing a mutation in DPAGT1),[10] have been reported. However, all of these models show embryonic lethality, making it difficult to use them to study the effects of CDG-I. Several studies have reported that patients with mutations in Alg13 can survive for 1 year to several decades.[1,11,12] Therefore, it is conceivable that animal models with Alg13 gene mutations may also survive past the embryonic stage.

In this study, we used the powerful clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system to establish a new mouse model for CDG-I involving the knock-out (KO) of Alg13 transcript variant 1. The study had two objectives: (i) designing a functional CRISPR/Cas9 system to modify Alg13 expression in mice, and (ii) identifying the Alg13 KO phenotype.

Materials and methods

Animals

All mice (C57BL/6) were kept at the Laboratory Animal Center of Shanghai Jiao Tong University, China (license No. SYXK (Hu) 2018-0028). Mice were housed in standard cages and maintained on a 12-hour light/dark cycle with food and water. All surgical procedures were performed in accordance with the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) and were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University (A2016084) on October 8, 2016.

Designing a CRISPR/Cas9 system to target mouse Alg13

Guide RNAs were designed to target the first and fourth exons of the murine Alg13 variant 1. Two sequences were chosen and used to construct single guide (sg) RNA expression plasmids based on the pUC57-sgRNA expression vector. In brief, we digested pUC57-sgRNA with BsaI and then ligated a pair of oligonucleotides containing the customized 20nt targeting sequences and BsaI restriction sites into the vector backbone, thus generating pAlg13-1 and pAlg13-2. The sequences of the oligonucleotides are shown in Additional Table 1, http://links.lww.com/JR9/A13.

Generating the Alg13 mutant mouse

The Cas9 expression plasmid was linearized with AgeI and used as a template for in vitro transcription with the T7 Ultra Kit (Ambion, Austin, TX, USA, Product Number: AM1345). SgRNA expression plasmids were linearized with DraI and used as templates for in vitro transcription using the MEGA shortscript Kit (Ambion, Product Number: AM1354). Transcribed Cas9 mRNA and sgRNA were purified using the MEGA clear Kit (Ambion, Product Number: AM1908).[13]

Fertilized mouse eggs were microinjected as described previously.[14] In brief, C57BL/6 female mice were injected with 5U of pregnant mare serum gonadotropin (PMSG, Sigma, St. Louis, MO, USA, Product Number: G4877), and 48 hours later injected, 5U of human chorionic gonadotropin (HCG, Sigma), after which they were immediately mated with male mice. Zygotes were harvested the next day and cultured in M16 medium (Sigma, Product Number: M7297) at 37°C for 30 minutes. Zygotes were injected with a mixture of 20 ng/μL Cas9 mRNA and 10 ng/μL of a sgRNA mix containing 20 ng/μL AlgE1 and 20 ng/μL AlgE4, which target two independent loci. The mixtures were microinjected into the cytoplasm of fertilized eggs using an Eppendorf microinjection system (Eppendorf, Saxony, Germany). After 30 minutes of culture in M16 medium, 15 to 25 injected zygotes were then transferred to a pseudo pregnant mouse to be carried to delivery.

Identification and analysis of the mice carrying Alg13 mutations

A total of six recipient mice delivered 6 pups (the founder mice (F0): 1# (male), 2# (female), 3# (female), 4# (female), 5# (male) and 6# (female)). Genomic DNA was extracted from the tails of the 7-day-old pups using a standard protocol. A T7 Endonuclease 1 (T7EN1) cleavage assay was performed as described previously.[15] Briefly, polymerase chain reaction (PCR) was performed as the following conditions: 95°C for 5 minutes, 35 cycles of 95°C for 30 seconds, 63°C for 30 seconds, 72°C for 40 seconds, then 72°C for 10 minutes. The PCR products were purified using a PCR clean-up kit (Axygen, Silicon Valley, CA, USA, Product Number: AP-PCR-50) and then denatured and re-annealed in NEB Buffer 2 (NEB, USA, Product Number: #B7202S) prior to digestion with T7EN1 (NEB, Product Number: M0302L) for 40 minutes and separation by 1% agarose gel electrophoresis. T7EN1 cleavage bands indicated modification of the targeting site.

The PCR products were sent for sequencing analysis to detect mutations. The sequences of the primers used to amplify the sgRNA target fragments from Alg13 exons 1 and 4 are listed in Additional Table 2, http://links.lww.com/JR9/A13. If multiple peaks were detected by sequencing, we sub-cloned the PCR products into T-vectors for sequencing analysis. If no wild-type allele was observed by sequencing, we assumed the mice had bi-allelic mutations. If a wild-type allele was detected, we assumed the mice harbored monoallelic mutations. If more than two genotypes were detected, we assumed the mice had mosaicism.

Characterization of the mutant mice

Homozygous second filial generation (F2) offspring were produced by mating. Behavioral characterization of seizures was performed using Racine's scale (RS) based on the behaviors observed during a seizure. There are 5 levels of epilepsy, depending on the severity of the seizure:-mouth and facial movements, head nodding, forelimb clonus, rearing and rearing and falling.[16] Epilepsy with significant behavioral manifestations classified as grade 3 and above is referred to as “convulsions”. To determine whether seizures detectable using the RS stage were a pathological consequence of Alg13 mutations, behavioral videos of the F2 mice were analyzed during the 4- to 10-week period. Only seizures graded as Racine stage 4 or higher were analyzed. Wild-type male F2 offspring were used as the control group.

Western blot

The cells were collected and then lysed with RIPA buffer (Yeasen, Shanghai, China, Product Number: 20101ES60), after incubation on ice for 30 minutes, the supernatant was collected by centrifugation at 12,000 r/min 4°C for 10 minutes. Following SDS-PAGE (70 V for 30 minutes, then 120 V for 1 hour), proteins were transferred to PVDF membranes (100 V for 90 minutes). After blocking in 5% non-fat milk for 2 hours at room temperature, the PVDF membranes were incubated with primary antibodies (rabbit anti-Alg13, Catalog Number: 20810-1-AP, Proteintech, 1:1000; goat anti-β-actin antibody, Catalog Number: ab8229, Abcam, 1:1000) at 4°C overnight. After three 10-minute washes with TBST, the membranes were incubated with secondary antibodies at room temperature for 2 hours. Finally, the labeled proteins were detected with super ECL detection reagent (Yeasen, Product Number: 36208ES60).

Statistical analysis

Statistical analyses were conducted using IBM SPSS Statistics 20.0 software (IBM, Armonk, NY, USA). The litter size and the number of mice that were positive for epilepsy based on the RS were analyzed. One-way analysis of variance was used to compare differences between groups. P < 0.05 was considered statistically significant.

Results

Design of CRISPR/Cas9 plasmids

The CRISPR/Cas9 system was used to knock out mouse Alg13 (Fig. 1A). A pair of sgRNAs, G1 and G4 (Fig. 1B), were chosen to construct the sgRNA plasmids. Cas9 sequences were expressed using the pST1374-Cas9-N-NLS-flag-linker plasmid (AddgeneID, Catalog Number: 44758).[17] The G1 sgRNA targets exon 1 of Alg13, while the G4 sgRNA targets exon 4, to enable the intervening sequence to be knocked out. Knocking out this sequence created 2 frame shift mutations that affected the downstream portion of the mouse gene.

Figure 1
Figure 1:
Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system designed to target asparagine-linked glycosylation 13 homolog (Alg13). (A) Schematic of DNA binding sites and G1 single guide (sg) RNA sequence in Alg13 exon 1. (B) Sites and sequences of sgRNA G1 and G2 in Alg13. PAM=protospacer adjacent motif.

Generation of Alg13 KO mice

To produce Alg13 KO mice, sgRNA transcripts and Cas9 mRNA were injected into mouse zygotes. A total of 125 zygotes were transferred into 6 pseudo pregnant mice, resulting in the birth of 6 pups. Transgenic founder animals were identified by the T7EN assay and PCR amplification and sequencing. Three transgenic founders (2#, 4#, and 5#) were shown to carry frame shift mutations in exon 4 of Alg13 transcript variant 1 (Fig. 2A). In addition, sequencing analysis showed that these mice were homozygous for this mutation (Fig. 2B). The genotypes of these founders are shown in Figure 2C: 2# contained a 54-bp deletion; 4# contained two 4-bp deletions in different alleles; and 5# contained two 5-bp deletions in different alleles.

Figure 2
Figure 2:
Identification of asparagine-linked glycosylation 13 homolog (Alg13) mutations in candidate mice. (A) Cas9 detection: sgRNA-mediated site-specific cleavage of endogenous G4 by T7EN1. Polymerase chain reaction (PCR) products from the targeted G4 fragment in six founder mice (#1–6) were subjected to a T7 Endonuclease 1 (T7EN1) cleavage assay. (B) Sequencing analysis of three mice. The modified site is indicated with an arrow. (C) DNA sequences of Alg13 exon 4 in founders #2, #4, and #5. PCR products that were successfully cleaved by T7EN1 were cloned and sequenced. Deletions (–) are shown to the right of each allele. Cas9=clustered regularly interspaced palindromic repeats-associated protein 9, M = marker, sgRNA = single guide RNA, WT = wild type.

Characterization of mice carrying Alg13 mutations

Patients with Alg13 mutations often show symptoms such as eating disorders, mental retardation, visual retardation and early-onset epilepsy.[1] The F2 homozygous mice generated in this study exhibited similar phenotypes, including feeding problems (Fig. 3A–C) and developmental block (Fig. 3D). Compared with wild-type mice, the Alg13 transcript variant 1 mutant mice had a smaller average litter size (Fig. 3E). Western blot analysis demonstrated that Alg13–5nt mice expressed lower levels of Alg13 than wild-type mice (Fig. 3F). These results suggest that Alg13 mutant mice could be used as a model to study the developmental disorders caused by Alg13 mutation.

Figure 3
Figure 3:
Mice carrying asparagine-linked glycosylation 13 homolog (Alg13) mutation. (A–C) Mice carrying the Alg13 mutation exhibited feeding difficulties. (A) One pup died at day 1. (B) Eating difficulties were apparent and some offspring died at about 2 weeks (C). (D) Some of the fetuses carrying Alg13 mutation that died at embryonic day 15. Blue arrows indicate the dead embryos. (E) The average litter size of an Alg13 –5nt/–5nt female × Alg13 –5nt male cross. (F) Western blot analysis of Alg13 –5nt and wild type (WT) mice.

Alg13 mutant mice exhibit spontaneous seizures as defined by the RS

In one of the transgenic founder mice (5#, male), spontaneous seizures were observed at 23 weeks. We selected 24 F2 offspring with the same genotype as this founder, and examined their behavior (male, 4 wild-type mice and 17 Alg13–5nt mice). As shown in Figure 4A (a–c), spontaneous seizures graded as Racine stage 5 were observed in 4 of the 17 (23.5%) male Alg13–5nt mice (as shown in Additional Videos 1–4, http://links.lww.com/JR9/A14, http://links.lww.com/JR9/A15, http://links.lww.com/JR9/A16, http://links.lww.com/JR9/A17). The remaining 8 (47%) male Alg13–5nt mice showed mild seizures graded as Racine stage 3 (as shown in Additional Video 5, http://links.lww.com/JR9/A18), whereas control mice did not exhibit any seizures (Fig. 4B). These results strongly suggest that mutations in Alg13 induce epilepsy in mice.

Figure 4
Figure 4:
Epilepsy in the asparagine-linked glycosylation 13 homolog gene (Alg13) modified mice. (A) Two mice were confirmed to have stage 5 seizures involving jumping (a), tail-raising (b) and falling (c). (B) Percentage of male Alg13 –5nt animals with seizures. SRS = seizures.

Discussion

Alg13 has been identified as a de novo mutation associated with epileptic encephalopathies.[11] A boy carrying an ALG13 280A>G variant exhibited polymorphic seizures, atrophy of the optic nerve, susceptibility to infection, hepatomegaly, and swelling of the eyelids and extremities.[5] Moreover, four boys reported to carry the novel missense ALG13 variant 3221A>G exhibited non-syndromic X-linked intellectual disability disorder, although no further clinical information was provided.[18] Additionally, five girls with early onset severe epilepsy and intellectual disability were found to have a c.320A>G variant in ALG13.[1,11,12] Such cases suggested the possibility of creating a mouse model of epilepsy by mutating by Alg13.

Alg13 variants have also been identified as being causative for CDG-Is. For example, Timal et al[5] reported an ALG13 missense mutation (c.280A>G; p.Lys94Glu) in a boy with CDG and a metabolic signature consistent with a defect in N-glycosylation type Is (also known as CDG-Alg13). The boy was confirmed to be hemizygous for the ALG13 mutation, and died at the age of 1 year. Transferrin isoform analysis, isoelectric focusing with affinity chromatography and mass spectrometry were used to detect whether a girl with an ALG13 variant (c.320A>G) had CDG, and while the results were negative, enzyme activity was undetectable.[1] There is no clear explanation for the differing results between these two cases, although it is possible that ALG13 expression differs in different tissues (and perhaps specific for certain tissues) and that the need for the enzyme is different in different contexts.

CDG-I is always associated with severe disease. However, although CDGs have been known of for decades, their pathophysiology is largely unexplored,[4,19] and few analyses of these patients have been performed.[20–22] Patients with CDG-I often exhibit a variety of phenotypes, most of which are related to neurological abnormalities, such as epilepsy, psychomotor retardation and hypotonia. In addition, abnormal liver, kidney, coagulation and endocrine functions, as well as deformities, have also been observed.[5] Furthermore, the clinical characteristics of defects caused by different glycosylation pathways are often similar, so it can be challenging to effectively distinguish between the effects of different defects. In general, the genetic defects associated with CDGs only lead to diagnostic clinical features in a few cases, such as the muscular dystrophy caused by mutations in dolichyl-phosphate mannosyltransferase polypeptide 3-CDG. Moreover, the large number of genes that may be involved in protein N-glycosylation makes it extremely difficult to identify the cause of the defect by direct sequencing. Therefore, animal models provide a means of studying the whole-body effects of CDG-I.

Several animals models have been developed to investigate human CDG.[22] Previous reports have shown that mutations in genes encoding most of the activities involved in N-glycan synthesis are embryonic lethal.[4,6–10,23] Furthermore, no live mouse models are available for different types of CDG associated with different steps in the metabolic pathway (Fig. 5 and Additional Table 2, http://links.lww.com/JR9/A13).[9,22,24,25] Because mutations in several CDG-I genes have been reported to cause epilepsy (Fig. 5),[4,26–31] we attempted to create a mouse model of epilepsy by knocking out or knocking down Alg13 transcript variant 1.

Figure 5
Figure 5:
Genes associated with epilepsy in CDG-I. Previous reports have shown that mutations in several genes, indicated here with red circles, have been reported to cause epilepsy in the context of CDG-I.[4,26–31] Several mutations in genes encoding proteins involved in N-glycan synthesis are embryonic lethal, and are indicated here with “×”.[4,6–10,23] CDG-I = congenital disorders of glycosylation type I.

In summary, we successfully generated an Alg135nt/-5nt male mouse model that experiences recurrent seizures and could be used as an animal model for studying epilepsy induced by Alg13 mutation.

Acknowledgments

We thank Professor Xingxu Huang, Professor Bin Shen at Shanghai Key Laboratory of Reproductive Medicine in China, for their kind help during designing CRISPR/Cas9 system.

Author contributions

BY and GZ performed the experiment and wrote the manuscript. SgL, XC, QF and YZ analyzed the results. YL, StL and JW approved the final manuscript.

Financial support

This study was partially supported by the National Natural Science Foundation of China (Nos. 81720108017, 81370675 and 81421061); Scientific Research Program of Ningxia Medical University, China (No. 2002080101).

Institutional review board statement

All surgical procedures were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University (A2016084) on October 8, 2016.

Conflicts of interest

The authors declare that there are no conflicts of interest regarding the publication of this paper.

References

[1]. Smith-Packard B, Myers SM, Williams MS. Girls with seizures due to the c.320A>G variant in ALG13 do not show abnormal glycosylation pattern on standard testing. JIMD Rep 2015;22:95–98.
[2]. Gao XD, Tachikawa H, Sato T, et al Alg14 recruits Alg13 to the cytoplasmic face of the endoplasmic reticulum to form a novel bipartite UDP-N-acetylglucosamine transferase required for the second step of N-linked glycosylation. J Biol Chem 2005;280:36254–36262.
[3]. Gao XD, Moriyama S, Miura N, et al Interaction between the C termini of Alg13 and Alg14 mediates formation of the active UDP-N-acetylglucosamine transferase complex. J Biol Chem 2008;283:32534–32541.
[4]. Stanley P. What have we learned from glycosyltransferase knockouts in mice? J Mol Biol 2016;428:3166–3182.
[5]. Timal S, Hoischen A, Lehle L, et al Gene identification in the congenital disorders of glycosylation type I by whole-exome sequencing. Hum Mol Genet 2012;21:4151–4161.
[6]. Thiel C, Lübke T, Matthijs G, et al Targeted disruption of the mouse phosphomannomutase 2 gene causes early embryonic lethality. Mol Cell Biol 2006;26:5615–5620.
[7]. DeRossi C, Bode L, Eklund EA, et al Ablation of mouse phosphomannose isomerase (Mpi) causes mannose 6-phosphate accumulation, toxicity, and embryonic lethality. J Biol Chem 2006;281:5916–5927.
[8]. Ioffe E, Stanley P. Mice lacking N-acetylglucosaminyltransferase I activity die at mid-gestation, revealing an essential role for complex or hybrid N-linked carbohydrates. Proc Natl Acad Sci U S A 1994;91:728–732.
[9]. Sung YH, Baek IJ, Seong JK, et al Mouse genetics: catalogue and scissors. BMB Rep 2012;45:686–692.
[10]. Marek KW, Vijay IK, Marth JD. A recessive deletion in the GlcNAc-1-phosphotransferase gene results in peri-implantation embryonic lethality. Glycobiology 1999;9:1263–1271.
[11]. Epi KC, Epilepsy Phenome/Genome P, Allen AS, et al De novo mutations in epileptic encephalopathies. Nature 2013;501:217–221.
[12]. Michaud JL, Lachance M, Hamdan FF, et al The genetic landscape of infantile spasms. Hum Mol Genet 2014;23:4846–4858.
[13]. Ma Y, Shen B, Zhang X, et al Heritable multiplex genetic engineering in rats using CRISPR/Cas9. PLoS One 2014;9:e89413.
[14]. Cheng Y, An LY, Yuan YG, et al Hybrid expression cassettes consisting of a milk protein promoter and a cytomegalovirus enhancer significantly increase mammary-specific expression of human lactoferrin in transgenic mice. Mol Reprod Dev 2012;79:573–585.
[15]. Hwang WY, Fu Y, Reyon D, et al Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 2013;31:227–229.
[16]. Racine RJ. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysiol 1972;32:281–294.
[17]. Shen B, Zhang J, Wu H, et al Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res 2013;23:720–723.
[18]. Bissar-Tadmouri N, Donahue WL, Al-Gazali L, et al X chromosome exome sequencing reveals a novel ALG13 mutation in a nonsyndromic intellectual disability family with multiple affected male siblings. Am J Med Genet A 2014;164A:164–169.
[19]. Clerc F, Reiding KR, Jansen BC, et al Human plasma protein N-glycosylation. Glycoconjugate J 2016;33:309–343.
[20]. Ferreira V, Brionesb P, Vilasecac MA. Congenital disorders of glycosylation (CDG): from glycoproteins to patient care. In: The Royal Society of Chemistry. Carbohydrate Chemistry 2012;124–155.
[21]. Schnaar RL. Glycans and glycan-binding proteins in immune regulation: A concise introduction to glycobiology for the allergist. J Allergy Clin Immunol 2015;135:609–615.
[22]. Thiel C, Körner C. Mouse models for congenital disorders of glycosylation. J Inherited Metab Dis 2011;34:879–889.
[23]. Akama TO, Nakagawa H, Wong NK, et al Essential and mutually compensatory roles of {alpha}-mannosidase II and {alpha}-mannosidase IIx in N-glycan processing in vivo in mice. Proc Natl Acad Sci U S A 2006;103:8983–8988.
[24]. Dwyer CA, Baker E, Hu H, et al RPTP(/phosphacan is abnormally glycosylated in a model of muscle-eye-brain disease lacking functional POMGnT1. Neuroscience 2012;220:47–61.
[25]. Freeze HH, Eklund E, Cummings RD, Pierce JM. Chapter 17 -Introduction to human glycosylation disorders. In: Handbook of Glycomics. San Diego: Academic Press. 2010:431–464.
[26]. Aeby A, Prigogine C, Vilain C, et al RFT1-congenital disorder of glycosylation (CDG) syndrome: a cause of early-onset severe epilepsy. Epileptic Disord 2016;18:92–96.
[27]. Barba C, Darra F, Cusmai R, et al Congenital disorders of glycosylation presenting as epileptic encephalopathy with migrating partial seizures in infancy. Dev Med Child Neurol 2016;58:1085–1091.
[28]. Dörre K, Olczak M, Wada Y, et al A new case of UDP-galactose transporter deficiency (SLC35A2-CDG): molecular basis, clinical phenotype, and therapeutic approach. J Inherited Metab Dis 2015;38:931–940.
[29]. Morava E, Tiemes V, Thiel C, et al ALG6-CDG: a recognizable phenotype with epilepsy, proximal muscle weakness, ataxia and behavioral and limb anomalies. J Inherited Metab Dis 2016;39:713–723.
[30]. Regal L, van Hasselt PM, Foulquier F, et al ALG11-CDG: Three novel mutations and further characterization of the phenotype. Mol Genet Metab Rep 2014;2:16–19.
[31]. Vianey-Saban C, Acquaviva C, Cheillan D, et al Antenatal manifestations of inborn errors of metabolism: biological diagnosis. J Inherited Metab Dis 2016;39:611–624.
Keywords:

Alg13; CRISPR/Cas9; epilepsy; mouse; mutant

Supplemental Digital Content

Copyright © 2020 The Chinese Medical Association. Published by Wolters Kluwer Health, Inc.