Clinical and genetic characterization of Congenital disorders of glycosylation in 20 Chinese patients: novel variants and genotype-phenotype correlations

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These disorders can affect multiple organs, leading to a broad spectrum of symptoms that vary among different CDG subtypes and between individuals with same type of CDG. This study aimed to investigate the genetic variants, molecular etiologies, and clinical features of 20 Chinese patients diagnosed with CDG. Results: Using whole-exome sequencing (WES), functional prediction tools, Sanger sequencing, and segregation analysis, we identified variants in several genes: ALG2 (3 patients), DPM2 (3 patients), PMM2 (3 patients), and ALG13 (2 patients). Additionally, variants in COG5 , COG6 , MOGS , DPM3 , ALG1 , ALG3 , ALG11 , SSR4 and SLC35A2 each were observed in single case. In total, 28 distinct variants were identified, 11 of which were previously unreported. Genotype-phenotype correlations revealed notable findings: variants in the N-terminus of ALG2 before the intramembrane domain were associated with congenital myasthenic syndromes (CMS), whereas those in the C-terminus caused ALG2-CDG; DPM2-CDG patients with variants in transmembrane region 1 exhibited more severe phenotypes; male patients with hemizygous variants in SLC35A2 demonstratedmilder phenotypes compared to those with mosaic variants. Conclusions: This findings expand the spectrum of known clinical presentations and genetic variants in CDG, and establish possible genotype-phenotype correlations of several pathogenic genes, emphasizing the need for functional studies to unravel the underlying mechanisms. Congenital disorders of glycosylation genetic variants genotype-phenotype correlation whole-exome sequencing novel variants Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Congenital disorders of glycosylation (CDG), are a heterogeneous group of inherited metabolic disorders caused by defects in glycosylation pathways ( 1 – 2 ). Glycosylation is the predominant form of post-translational modification for proteins and lipids to by transferreing specific amino acid residues by glycosyltransferases, and plays an important role in protein folding, immune recognition, and other physiological processes( 3 – 4 ). Glycosylation is ubiquitous, occurring in every cell and organism, with over half of all cellular proteins undergoing glycosylated. Consequently, CDG often present highly diverse clinical presentations and involve multisystems( 5 – 6 ). Typical clinical features of CDG include neurological and developmental disabilities, such as psychomotor retardation/cognitive impairment, epilepsy, hytotonia, and ataxia. Other systems or organs, including hepatic, gastrointestinal, hormonal, and immune systems as well as the heart, eyes, and skeleton, are usually affected. In addition, the severity of symptoms ranges widely, from perinatal death or miscarriage to mild adult-onset forms( 7 ). Since the first CDG, PMM2-CDG, was discovered in 1980, the field has been expanding rapidly with the application of next-generation sequencing in clinical practice, with over 160 subtypes identified, which enhances our understanding of these disorders. In this study, we analyzed 20 patients from China, with all probands evaluated using whole-exome sequencing (WES). Among the cohort, we identified 28 variants in several genes: ALG2 (3 cases), DPM2 (3 cases), PMM2 (3 cases) and ALG13 (2 cases), with the remaining patients carrying variants in COG5 , COG6 , MOGS , DPM3 , ALG1 , ALG3 , ALG11 , SSR4 and SLC35A2 , 11 of which are novel and have not been reported. These findings provide valuable insights into CDG genetic diversity and offer foundational clues for future diagnosis and therapeutic advancements. Materials and Methods Study subjects Patients with initial symptoms such as seizures, growth retardation, delayed cognitive and motor development, and visual or speech impairments were investigated. Patients with symptoms attributed to environmental and non-genetic factors were excluded. Detailed medical and clinical data, including family history, MRI neuroimaging, electroencephalography (EEG), and other relevant examinations, were collected. This study included 20 affected individuals suspicious or diagnosed of having CDG, along with their healthy family members. All patients were born into families with healthy parents and exhibited normal at birth. Six families underwent prenatal diagnosis following a confirmed diagnosis. Six families underwent prenatal diagnosis following a confirmed CDG diagnosis. The study was approved by the ethics committee of the Institutional Review Board (IRB) of Wuhan Children's Hospital, Tongji Medical College, Huazhong University of Science & Technology, with the approval code 2021R060-E1. Written informed consent was secured from all participants whose data are presented individually in accordance with the principles outlined in the Declaration of Helsinki. Whole-exome sequencing and data analysis Whole WES and subsequent data analysis were conducted with the help of the third party medical laboratory (Chigene Lab, Beijing China). For WES, the target DNA fragments were enriched by hybridization to construct an exome library (IDT The xGen Exome Research Panel v1.0). High-throughput sequencing was performed using an Illumina NovaSeq 6000 sequencer. Single-nucleotide and indel variants were identified using the Genome Analysis Toolkit (GATK). Paired-end alignment was performed using the Burrows-Wheeler Aligner (BWA). Moreover, ANNOVAR software annotated and filtered the variants. SNPs and indels were filtered and screened according to sequence depth and mutation quality. The variants were annotated using the OMIM, ClinVar and HGMD databases. Candidate variants were confirmed by Sanger sequencing using self-designed primers in the patients and their parents. To predict the pathogenicity of the variant, we used several bioinformatics software and tools. At first, variants were preferentially selected for further analysis and validation with their minor allele frequency (MAF)<0.01 in the ExAc browser (https://exac.broadinstitute.org) and gnomAD (https://gnomad.broadinstitute.org) database . Then, single nucleotide variants(SNV) and short indel candidates were identified. The SIFT utilities were used to forecast the change in protein structure. Conservation of each amino acid change was calculated using PhyloP2 and MutationTaster (http://www.mutationtaster.org/) algorithms were used to predict the effects of variants on protein function. All variants were named according to the guidelines of the Human Genome Variation Society (http://www.hgvs.org/) and were described and classified based on the standard guidelines of American College of Medical Genetics and Genomics(ACMG)(8). Genotype-phenotype associations The novel candidate variants in this study or previously reported mutations to cause CDG disease was summarized and reviewed and adapted to the clinical symptoms of each patient. They were also determined using several databases such as Human Gene Mutation Database, PubMed (https://www.ncbi.nlm.nih.gov/pubmed/), and Online Mendelian Inheritance in Man (https://omim.org/) to analysis the association between genotype and phenotype. Results Overall characteristics and molecular diagnosis This study included 20 CDG patients (13 males and 7 females) from 18 non-consanguineous families (Fig.1 and Table 1). Clinical symptoms were presented for 20 cases and cataloged using Human Phenotype Ontology (HPO) terms (Table 1). The overall median age of symptom onset was 6.0 months (range: 1.0–48.0 months), and the median age at diagnosis was 1.45 years (range: 4 months to 20 years). The most common symptoms were developmental delay (100%, 19/19), followed by facial dysmorphia (64.7%, 11/17), hearing or ophthalmological problems (52.9%, 9/17), abnormal liver function (52.6%, 10/19) and seizures(50%) (Fig. 2). By using WES, we identified 28 disease-causing variants in 13 genes among the 20 patients. Among these variants, 17 previously reported and 11 novel variants. A summary of detected mutations is provided in Table 2. The patients were followed from 6 months to 5 years in outpatient of Wuhan children’s hospatal. During this time, two patients died due to recurrent intractable epilepsy associated with their diseases (P7 and P14). Prenatal diagnosis was performed in six families (F2, F3, F7, F12, F13 and F14) using amniotic fluid cells. Patients with variants in the ALG2 gene Variants in the ALG2 gene (NM_003087) are associated with ALG2-CDG, also known as CDG Ii (OMIM#607906), which is characterized by neurological symptoms such as convulsive syndrome, epilepsy, axial hypotonia, mental and motor regression (9). Additionally, ALG2 variants can cause congenital myasthenic syndrome type 14 (ALG2-CMS, OMIM#616228), which presents within the first decade of life, progresses slowly (10). In this study, three patients (P5, P6 and P7) from 2 families with compound heterozygous ALG2 variants were included. The initial symptom of these 3 patients was epileptic seizures. All of them exhibited seizures, mental and developmental regression, severe speech defects, and abnormal liver function. Genetic analysis identified compound heterozygous variants (c.422A>T (p.D141V) and c.751C>T (p.R251C)) in P5, and variants (c.751C>T and c.935_937del (p.L312del)) in P6 and P7. According to standard guidelines of ACMG, c.751C>T was classified as LP, while c.422A>T and c.935_937del as VUS. The potential effects of these variants were predicted using a three-dimensional structural model, indicating Protein stability being reduced compared to the wild type (lower panel in Fig. 3). Based on typical clinical manifestations and genetic findings, these patients were diagnosed with ALG2-CDG. Patients with variants in the DPM2 gene DPM2-CDG (OMIM#615042) is an extremely rare subtype of CDG.In this study, P12 presented with seizures, followed by neurological and motor developmental delay, and elevated CK levels. Genetic analysis identified a homozygous variant c.176T>G in exon 4 of DPM2 (NM_003863.4), inherited from heterozygous parents. Two additional cases, P13 and P14, had been previously reported (11). Together with our case in this study, only six cases from three families have been identified (12-13). Patients with variants in SSR4 gene SSR4-CDG is a rare and relatively mild subtype of CDG (also known as CDG Iy), predominantly affecting males (14). Key clinical features include developmental delay, speech delay, intellectual disability, muscular hypotonia, microcephaly, skeletal abnormalities, and distinct facial features (15). In this study, we identified a hemizygous spanning exons 4 to 6 of SSR4 in P18, inherited from his healthy mother, in a 16-month-old boy. He was born at 39 weeks following a pregnancy complicated by intrauterine growth retardation,.and present with global developmental delay, speech delay, cognitive impairment, hypotonia, frbrile seizures, feeding difficulty, and mild facial dysmorphisms (large ears, smooth and long philtrum, abnormal eye distance, prominent nose). A literature review revealed 11 point variants and 7 fragment deletions in SSR4 from 23 SSR4-CDG patients (15)(Fig.4B). High degree of phenotypic heterogeneity across individuals was found and no clear genotype-phenotype correlation has been established. Patients with variants in SLC35A2 gene SLC35A2-CDG, an X-linked dominant disorder, typically manifests during infancy. The condition is characterized by microcephaly, psychomotor development delay, epileptic seizures, and hypotonia (16). Male patients usually carry mosaic variants (17). A hemizygous variant (c.902C>T) in was SLC35A2 was identified in P20. He was a 35-month-old boy with short stature (<-3sd), mild language and motor developmental delay, mild facial dysmorphisms, pectus carinatum, and elevated aspartate transaminase (AST) levels. A literature review identified four male cases with hemizygous variants in SLC35A2 (18-20), and their clinical features included short stature, abnormal liver function, language delay, intellectual disability, mild facial abnormalities, skeletal deformities and epileptic seizures (19). In addition, male patients with mosaic SLC35A2 variants were reported to exhibit more severe symptoms, including epileptic encephalopathy or drug-resistant focal epilepsy (21-22). Based on these findings, we propose that male patients with hemizygous SLC35A2 variants generally present with milder phenotypes compared to those with mosaic variants. Patients with variants in the PMM2 gene PMM2-CDG (CDG Ia, OMIM#212065) is the most common CDG with more than 1000 cases recorded worldwide (23-24). Three patients (P8, P9, and P10) presented with developmental delay and hypotonia. They also had abnormal liver functions, evidenced by elevated aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Brain MRI showed cerebellar atrophy and white matter demyelination. Compound heterozygous variants in PMM2 (NM_000303.3) were identified in all three cases. The variant c.406T>G (p.C136G) in P9 was first reported in this work and classified as LP (PM1, PM2, PP2, PP3) according to ACMG guidelines. Patients with variants in the ALG13 gene ALG13-CDG (OMIM#300884) is a rare X-linked CDG, which affects the N-linked glycosylation pathway (25). Affected individuals typically exhibit refractory seizures, psychomotor development delay, poor or absent speech, hypotonia, facial dysmorphism, and intellectual disability (26). Epileptic spasm is a common presenting symptom of ALG13-CDG. Two unrelated patients (P16 and P17) with infantile spasms associated with hypsarrhythmia on EEG as the initial symptom was identified (Table 1). Both patients currently exhibit delayed language and motor development. In addition, P16 has the characteristic of recurrent infections. Brain MRI showed a right choroidalcyst and widening of bilateral lateral fissures intracranial space. A de novo heterozygous variant c.320A>G (p.N107S) in ALG13 (NM_001099922.3), a known hotspot pathogenic variant, was identified in P16. In P17, a hemizygous variant (c.999G>C, p.K333N) was found and classified as a VUS in patient 16, inherited from the patient's affected mother. Patients with variants in COG5 and COG6 genes The COG (conserved oligomeric Golgi) complex is composed of 8 subunits, including lobe A (from subunit COG1 to COG4) and lobe B (from subunit COG5 to COG8)(27). Genetic defect seven subunits (all but COG3) disrupts the function of the COG complex and have been reported to be associated with different types of CDG. In our study, two patients (P1 and P2) had genetic variants in COG5 (c.1039C>T, c.928+3A>G) and COG6 (c.1843C>T, c.428G>T), respectively, an the patient P2 had been reported before (28). Their clinical phenotypes were shown in Table 1 and similar to previously reported cases. Patients with variants in ALG1 gene ALG1-CDG is an autosomal recessive disease with severe multiorgan involvement (OMIM 608540) caused by pathogenic variants in ALG1(29). The most common phenotypic manifestations are developmental delay, intellectual disability, failure to thrive, hypotonia, and epilepsy. P15, 1-year-boy, had compound heterozygous variants in ALG1 gene (NM_019109, c.328C>A, and c.863-2A>G), and these two variants were classified as LP and P according to ACMG guidelines. This boy had drug-resistant epilepsy, facial dysmorphisms and abnormal liver function. and died at 14 month old. Prenatal diagnosis was performed for this family when his mother was pregnant and they got a healthy baby. Discussion Despite global recognition, CDG is under reported in China, with limited case studies available. This is first comprehensive report from China to analyze 20 diagnosed CDG cases, highlighting clinical manifestations such as seizures, motor delays, facial dysmorphism, and growth retardation, supported by findings like abnormal MRI neuroimaging (e.g., cerebellar atrophy), elevated CK levels, and abnormal liver function. This study underscores the importance of integrating genomic tools and systematic evaluations to improve CDG diagnosis and characterization. A recent large cohort study of 280 individuals with genetic data consistent with a CDG diagnosis found that (s) developmental delay was the most frequent presenting symptom (77%), followed by hypotonia (56%) and feeding difficulties (42%) ( 30 ). Similarly, in our cohort, development delay was the most common initial symptom, observed in all individuals by the time of enrollment, followed by facial dysmorphism (64.7%), abnormal liver function (52.6%), and seizures (50%). The broad differential diagnosis associated with these symptoms, reflecting significant clinical heterogeneity, continues to pose a challenge for timely CDG diagnosis. We found 28 distinct variants, including 11 novel variants, in 13 different genes: ALG1 , ALG2 , ALG3 , ALG11 , ALG13 , COG5 , COG6 , MOGS , DPM2 , DPM3 , SSR4 , SLC35A2 , and PMM2 . In line with previously findings (38), the majority of variants were missense (78.5%), followed by nonsense (10.7%), splice site (7.1%), and frameshift deletions (3.6%) (Table 2 ). According to ACMG classification guidelines, 6 variants were classified as pathogenic (P), 8 as likely pathogenic (LP), and 14 as variants of uncertain significance (VUS). Autosomal recessive inheritance was observed in 80% (16/20) of cases and 20% (4/20) displayed X-linked inheritance. The most common variants were in genes associated with disorders of N-linked protein glycosylation, including PMM2 (3 patients from 3 families), ALG2 (3 from 2 families), ALG13 (2 patients), ALG1 ( 1 ), ALG3 ( 1 ), ALG11 ( 1 ), MOGS ( 1 ), and SSR4 ( 1 ), followed by genes causing disorders of dolichol metabolism (DPM2 and DPM3) and disorders of Golgi trafficking and transport ( COG5 , COG6 , and SLC35A2 ), which is consistent with previous report ( 30 ). This study significantly expands the current mutational spectrum of CDG, and also revealed considerable genetic heterogeneity, reflecting the complex molecular underpinnings of CDG. In this study, we conducted a correlation analysis between genotype and phenotype by retrieving all previously published cases, including our patients, for these genes associated with uncommon CDGs. ALG2 variants could develop ALG2-CDG or ALG2-CMS, by reviewing literature, a total of 9 cases of ALG2-CMS and 5 cases of ALG2-CDG were reported from 7 articles ( 9 – 10 , 31 – 34 ). Together with our study, 11 variants in ALG2 have been identified. we noticed that variants located before the intramembrane region (N-terminal, before 158aa) are associated with ALG2-CMS, while variants occurring after the intramembrane region are linked to ALG2-CDG, which presents with more severe clinical symptoms (Fig. 3 ). ALG2 takes part in the fourth and fifth step of lipid-linked oligosaccharide (LLO) synthesis, with two different mannosyltransferase activities. Specifically, Alg2 adds both α 1,3-and α1,6-mannose onto ManGlcNAc 2 –Pdol to form the trimannosyl core Man 3 GlcNAc 2 -PDol ( 30 ). The Alg2 conserved C-terminal EX7E motif, the N-terminal cytosolic tail, and 3G-rich loop motifs, are essential for these enzymatic activities, both in vitro and in vivo ( 35 ). Variants in these regions impair the enzyme’s function, which likely contributes to the more severe clinical manifestations observed in ALG2-CDG compared to the relatively milder phenotype of CMS caused by ALG2 variants. DPM2-CDG is an extremely rare form of CDG, and the main clinical manifestations of these patients include hypotonia, intractable epilepsy, muscle damage, elevated serum creatine kinase (CK) levels, and microcephaly ( 11 ). To date, including our cases, only 7 cases from four families have been identified ( 11 – 13 ). Our analysis revealed variants affecting the first transmembrane region or disrupting it were associated with more severe symptoms, and three patients with such variants died before the age of 3 years. In contrast, variants in the second transmembrane region resulted in less severe manifestations (Fig. 4 A). DPM2 contains two putative transmembrane domains (amino acid residues 11–31 and 49–69) ( 12 ). Variants in the first domain, such as Phe21 and Tyr23 substitutions, impair its ability to bind with DPM1 ( 36 ), suggesting that the first transmembrane region is crucial for maintaining the stability of the complex. SSR4-CDG is ultra-rare X linked, comparably mild subtype of CDG, presenting mostly in males. We reported a 16-month-old boy, who had most of the key symptoms of SSR4-CDG, developmental delay, speech delay, cognitive impairment, feeding difficulties, and muscular hypotonia. In addition, this patient had febrile seizure and ocular abnormality. However, he had no obvious microcephaly, and his facial feature is not as typical as described previously, which could be due to the young age. SLC35A2 , located on chromosome Xp11.23, encodes UGT-1 and is part of the SLC35 family of nucleotide sugar transporters (NSTs). De novo variants in this gene are associated with SLC35A2-CDG, often with epileptic encephalopathy as a prominent feature ( 16 ). Somatic SLC35A2 variants have also been reported in patients with focal seizures and suspected focal cortical dysplasia (FCD) ( 17 ). Furthermore, recent studies have linked brain somatic variations in SLC35A2 to mild malformation of cortical development with oligo dendroglial hyperplasia in epilepsy (MOGHE) ( 37 ). While SLC35A2-CDG typically shows a strong gender bias, with most cases occurring in females, there have been reports of male patients with a milder phenotype, including minor neurological involvement and growth deficiency ( 19 – 20 ). By reviewing literature, we postulate that males with hemizygous variants in SLC35A2 may present with a milder phenotype compared to those with mosaic variants in brain, although this requires further research. Our study has some limitations. First, due to the small number of patients enrolled, we were unable to analyze more specific genotype-phenotype correlations. The rarity of CDG and the limited screening of this condition in China may partly explain this limitation. Second, changes in glycan composition in patient serum were not analyzed by MALDI-TOF MS due to the lack of necessary equipment. In addition, we identified some variants of VUS, functional studies were not conducted. Further research with larger patient cohorts and long-term follow-up would enhance our understanding of these diseases, which is critical for elucidating pathogenic mechanisms, improving the genotype-phenotype correlation, refining genetic counseling, and developing new treatments. Conclusion We identified 28 disease-causing variants, including 11 novel variants, using WES in 20 CDG patients. Our findings expand the spectrum of known variants and their related clinical phenotypes in Chinese CDG patients and establish possible genotype-phenotype correlations of several genes. Abbreviations CDG: Congenital disorders of glycosylation VUS: variants of uncertain significance NST: nucleotide sugar transporters WES: whole-exome sequencing EEG:electroencephalography GATK: Genome Analysis Toolkit BWA: Burrows-Wheeler Aligner HPO: Human Phenotype Ontology FCD: focal cortical dysplasia LLO: lipid-linked oligosaccharide AST: aspartate aminotransferase ALT: alanine aminotransferase SNV: single nucleotide variants MAF: minor allele frequency Declarations Competing interests The authors declare that they have no competing interests. Ethics approval and consent to participate : t he study was approved by the ethics committee of the Institutional Review Board (IRB) of Wuhan Children's Hospital, Tongji Medical College, Huazhong University of Science & Technology, with the approval code 2020R006-E05. Written informed consent was secured from all participants whose data are presented individually in accordance with the principles outlined in the Declaration of Helsinki. Consent for publication： the consent for publication was obtained from our patients or their guardian. Availability of data and materials ： all data and materials are available from the corresponding on reasonable request. Competing interests : the authors declare that they have no competing interests. Funding : This work was supported by the grants of Hubei Provincial Natural Science Foundation Project (No.2023AFB893); Construction Project of Clinical Medical Research Center for Neurodevelopmental Disorders in Children in Hubei Province (HST2020-19); and Construction Project of Clinical Medical Research Center for Birth Defect Prevention and Treatment in Wuhan (WK[2023]123-4). Authors' contributions : Study concepts: Xuelian He, Shiqiong Zhou, Peiwei Zhao Study design: Peiwei Zhao, Li Tan Literature research: Qingjie Meng, Peiwei Zhao, Yanqiu Hu Clinical information collection: Shiqiong Zhou, Qingjie Meng, Yanqiu Hu Data acquisition: Qingjie Meng, Xiankai Zhang, Lei Zhang, Yanqiu Hu Data analysis/interpretation: Lei Zhang, Qingjie Meng, Xiankai Zhang, Li Tan Manuscript preparation: Xuelian He, Peiwei Zhao Manuscript editing: Xuelian He, Shiqiong Zhou Manuscript final version approval: Xuelinan He, Shiqiong Zhou Acknowledgement: we appreciate the patient participating in this study. References Francisco R, Brasil S, Poejo J, et al. Congenital disorders of glycosylation (CDG): state of the art in 2022. Orphanet J Rare Dis. 2023;18(1):329. Published 2023 Oct 19. doi:10.1186/s13023-023-02879-z Paprocka J, Jezela-Stanek A, Tylki-Szymańska A, Grunewald S. Congenital Disorders of Glycosylation from a Neurological Perspective. Brain Sci. 2021;11(1):88. doi:10.3390/brainsci11010088 Freeze HH. Genetic defects in the human glycome. Nat Rev Genet. 2006;7(7):537-551. doi:10.1038/nrg1894 Abu Bakar N, Lefeber DJ, van Scherpenzeel M. Clinical glycomics for the diagnosis of congenital disorders of glycosylation. J Inherit Metab Dis. 2018;41(3):499-513. doi:10.1007/s10545-018-0144-9 Marques-da-Silva D, Dos Reis Ferreira V, Monticelli M, et al. Liver involvement in congenital disorders of glycosylation (CDG). A systematic review of the literature. J Inherit Metab Dis. 2017;40(2):195-207. doi:10.1007/s10545-016-0012-4 Ng BG, Freeze HH. Perspectives on Glycosylation and Its Congenital Disorders. Trends Genet. 2018;34(6):466-476. doi:10.1016/j.tig.2018.03.002 Altassan R, Péanne R, Jaeken J, et al. International clinical guidelines for the management of phosphomannomutase 2-congenital disorders of glycosylation: Diagnosis, treatment and follow up. J Inherit Metab Dis. 2019 May;42(3):577. doi: 10.1002/jimd.12082 Richards S, Aziz N, Bale S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17(5):405-424. doi:10.1038/gim.2015.30 Thiel C, Schwarz M, Peng J, et al. A new type of congenital disorders of glycosylation (CDG-Ii) provides new insights into the early steps of dolichol-linked oligosaccharide biosynthesis. J Biol Chem . 2003;278(25):22498-22505. doi:10.1074/jbc.M302850200 Cossins J, Belaya K, Hicks D, et al. Congenital myasthenic syndromes due to mutations in ALG2 and ALG14. Brain . 2013;136(Pt 3):944-956. doi:10.1093/brain/awt010 Zhao P, Hu Y, Hu J, et al. Identification and characterization of a new variation in DPM2 gene in two Chinese siblings with mild intellectual impairment. Front Genet. 2023;14:930692. doi:10.3389/fgene.2023.930692 Barone R, Aiello C, Race V, et al. DPM2-CDG: a muscular dystrophy-dystroglycanopathy syndrome with severe epilepsy. Ann Neurol. 2012;72(4):550-558. doi:10.1002/ana.23632 Radenkovic S, Fitzpatrick-Schmidt T, Byeon SK, et al. Expanding the clinical and metabolic phenotype of DPM2 deficient congenital disorders of glycosylation. Mol Genet Metab. 2021;132(1):27-37. doi:10.1016/j.ymgme.2020.10.007 Ng BG, Raymond K, Kircher M, et al. Expanding the Molecular and Clinical Phenotype of SSR4-CDG. Hum Mutat. 2015;36(11):1048-1051. doi:10.1002/humu.22856 Johnsen C, Tabatadze N, Radenkovic S, et al. SSR4-CDG, an ultra-rare X-linked congenital disorder of glycosylation affecting the TRAP complex: Review of 22 affected individuals including the first adult patient. Mol Genet Metab. 2024;142(3):108477. doi:10.1016/j.ymgme.2024.108477 Ng BG, Buckingham KJ, Raymond K, et al. Mosaicism of the UDP-galactose transporter SLC35A2 causes a congenital disorder of glycosylation. Am J Hum Genet. 2013;92(4):632-636. doi:10.1016/j.ajhg.2013.03.012 Elziny S, Crino PB, Winawer M. SLC35A2 somatic variants in drug resistant epilepsy: FCD and MOGHE. Neurobiol Dis. 2023;187:106299. doi:10.1016/j.nbd.2023.106299 Vals MA, Ashikov A, Ilves P, et al. Clinical, neuroradiological, and biochemical features of SLC35A2-CDG patients. J Inherit Metab Dis. 2019;42(3):553-564. doi:10.1002/jimd.12055 Quelhas D, Correia J, Jaeken J, et al. SLC35A2-CDG: Novel variant and review. Mol Genet Metab Rep. 2021;26:100717. Published 2021 Jan 30. doi:10.1016/j.ymgmr.2021.100717 Kodríková R, Pakanová Z, Krchňák M, et al. N-Glycoprofiling of SLC35A2-CDG: Patient with a Novel Hemizygous Variant. Biomedicines. 2023;11(2):580. doi:10.3390/biomedicines11020580 Barba C, Blumcke I, Winawer MR, et al. Clinical Features, Neuropathology, and Surgical Outcome in Patients With Refractory Epilepsy and Brain Somatic Variants in the SLC35A2 Gene. Neurology. 2023;100(5):e528-e542. doi:10.1212/WNL.0000000000201471 Elziny S, Crino PB, Winawer M. SLC35A2 somatic variants in drug resistant epilepsy: FCD and MOGHE. Neurobiol Dis. 2023;187:106299. doi:10.1016/j.nbd.2023.106299 Muthusamy K, Perez-Ortiz JM, Ligezka AN, et al. Neurological manifestations in PMM2-congenital disorders of glycosylation (PMM2-CDG): Insights into clinico-radiological characteristics, recommendations for follow-up, and future directions. Genet Med. 2024;26(2):101027. doi:10.1016/j.gim.2023.101027 Vaes L, Rymen D, Cassiman D, et al. Genotype-Phenotype Correlations in PMM2-CDG. Genes (Basel). 2021;12(11):1658. doi:10.3390/genes12111658 Shah R, Eklund EA, Radenkovic S, et al. ALG13-Congenital Disorder of Glycosylation (ALG13-CDG): Updated clinical and molecular review and clinical management guidelines. Mol Genet Metab. 2024;142(2):108472. doi:10.1016/j.ymgme.2024.108472 Alsharhan H, He M, Edmondson AC, et al. ALG13 X-linked intellectual disability: New variants, glycosylation analysis, and expanded phenotypes. J Inherit Metab Dis. 2021;44(4):1001-1012. doi:10.1002/jimd.12378 Wang X, Han L, Wang XY, et al. Identification of Two Novel Mutations in COG5 Causing Congenital Disorder of Glycosylation. Front Genet. 2020;11:168. doi:10.3389/fgene.2020.00168 Ng BG, Shiryaev SA, Rymen D, et al. ALG1-CDG: Clinical and Molecular Characterization of 39 Unreported Patients. Hum Mutat. 2016;37(7):653-660. doi:10.1002/humu.22983 Lam C, Scaglia F, Berry GT, et al. Frontiers in congenital disorders of glycosylation consortium, a cross-sectional study report at year 5 of 280 individuals in the natural history cohort. Mol Genet Metab. 2024;142(4):108509. doi:10.1016/j.ymgme.2024.108509 Kämpf M, Absmanner B, Schwarz M, Lehle L. Biochemical characterization and membrane topology of Alg2 from Saccharomyces cerevisiae as a bifunctional alpha1,3- and 1,6-mannosyltransferase involved in lipid-linked oligosaccharide biosynthesis. J Biol Chem . 2009;284(18):11900-11912. doi:10.1074/jbc.M806416200 Martínez Duncker I, Mata-Salgado D, Shammas I, et al. Case report: Novel genotype of ALG2-CDG and confirmation of the heptasaccharide glycan (NeuAc-Gal-GlcNAc-Man2-GlcNAc2) as a specific diagnostic biomarker. Front Genet . 2024;15:1363558. doi:10.3389/fgene.2024.1363558 Monies DM, Al-Hindi HN, Al-Muhaizea MA, et al. Clinical and pathological heterogeneity of a congenital disorder of glycosylation manifesting as a myasthenic/myopathic syndrome. Neuromuscul Disord . 2014;24(4):353-359. doi:10.1016/j.nmd.2013.12.010 Papazoglu GM, Cubilla M, Pereyra M, et al. Mass spectrometry glycophenotype characterization of ALG2-CDG in Argentinean patients with a new genetic variant in homozygosis. Glycoconj J . 2021;38(2):191-200. doi:10.1007/s10719-021-09976-w Ehrstedt C, Liu WW, Frykholm C, Beeson D, Punga AR. Novel pathogenic ALG2 mutation causing congenital myasthenic syndrome: A case report. Neuromuscul Disord . 2022;32(1):80-83. doi:10.1016/j.nmd.2021.11.012 Li ST, Wang N, Xu XX, et al. Alternative routes for synthesis of N-linked glycans by Alg2 mannosyltransferase. FASEB J . 2018;32(5):2492-2506. doi:10.1096/fj.201701267R Maeda Y, Tomita S, Watanabe R, Ohishi K, Kinoshita T. DPM2 regulates biosynthesis of dolichol phosphate-mannose in mammalian cells: correct subcellular localization and stabilization of DPM1, and binding of dolichol phosphate. EMBO J . 1998;17(17):4920-4929. doi:10.1093/emboj/17.17.4920 Bonduelle T, Hartlieb T, Baldassari S, et al. Frequent SLC35A2 brain mosaicism in mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (MOGHE). Acta Neuropathol Commun . 2021;9(1):3. Published 2021 Jan 6. doi:10.1186/s40478-020-01085-3 Tables Table 1 and 2 are available in the Supplementary Files section. Supplementary Files Tab.doc Cite Share Download PDF Status: Published Journal Publication published 23 Dec, 2025 Read the published version in Orphanet Journal of Rare Diseases → Version 1 posted Editorial decision: Major revision 24 Apr, 2025 Reviewers agreed at journal 27 Feb, 2025 Reviewers invited by journal 24 Feb, 2025 Editor assigned by journal 13 Jan, 2025 First submitted to journal 10 Jan, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5807293","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":420294579,"identity":"d2aea402-fc8b-4696-be44-4ac1c260d74c","order_by":0,"name":"Peiwei Zhao","email":"","orcid":"","institution":"Genetics and Precision Medical Center, Wuhan Children's Hospital (Wuhan Maternal and Child Healthcare Hospital), Tongji Medical College, Huazhong University of Science \u0026 Technology","correspondingAuthor":false,"prefix":"","firstName":"Peiwei","middleName":"","lastName":"Zhao","suffix":""},{"id":420294580,"identity":"b98fea34-fb66-4e8a-8b04-7b98ffe3dbe3","order_by":1,"name":"Li Tan","email":"","orcid":"","institution":"Genetics and Precision Medical Center, Wuhan Children's Hospital (Wuhan Maternal and Child Healthcare Hospital), Tongji Medical College, Huazhong University of Science \u0026 Technology","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Tan","suffix":""},{"id":420294581,"identity":"6d96f415-1df9-4238-84f9-5deee6575023","order_by":2,"name":"Qingjie Meng","email":"","orcid":"","institution":"Clinical Laboratory, Wuhan Children's Hospital (Wuhan Maternal and Child Healthcare Hospital), Tongji Medical College, Huazhong University of Science \u0026Technology","correspondingAuthor":false,"prefix":"","firstName":"Qingjie","middleName":"","lastName":"Meng","suffix":""},{"id":420294582,"identity":"8111f8d6-9cb8-4f3d-94f3-fdeb9de00d2a","order_by":3,"name":"Lei Zhang","email":"","orcid":"","institution":"Genetics and Precision Medical Center, Wuhan Children's Hospital (Wuhan Maternal and Child Healthcare Hospital), Tongji Medical College, Huazhong Univeristy of Science \u0026 Technology","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Zhang","suffix":""},{"id":420294583,"identity":"f6495476-b9e5-40fb-b42f-81587e6dd4d2","order_by":4,"name":"Yufeng Huang","email":"","orcid":"","institution":"Genetics and Precision Medical Center, Wuhan Children's Hospital (Wuhan Maternal and Child Healthcare Hospital), Tongji Medical College, Huazhong University of Science \u0026 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He","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABD0lEQVRIiWNgGAWjYBACAzBZIMHAwN7YcPBDxQEw/8ADgloMgFp4Dh88LHHmAAMPSEsCYS1ALJGWfIC3DaKFAZ8Wc/YeM4kPBhby5gw5Bgck592Rsxc7/BBoi52cbgN2LZY9x9IkZxhIGO5sOGNwoHDbM2Me6TQDoJZkY7MDOBx2I/mYNI+BBOOGgz1AW7YdTuyRTgBpOZC4DZeW+w/bpP8YSNhvOMxjcIB3DkhL+gf8Wm4wH5MGhljihmNsCQd4G0BacgjYciYt2bLHQCJ5wxnmA4cljh025rmdU3AgwQCPX46fMbzxo6LOdsP9h80fP9QclmOfnb75w4cKOzlcWnABA9KUj4JRMApGwShABQDq02kLLi5R4gAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-6275-1219","institution":"Wuhan Children's Hospital (Wuhan Maternal and Child Healthcare Hospital), Tongji Medical College, Huazhong University of Science \u0026 Technology","correspondingAuthor":true,"prefix":"","firstName":"Xuelian","middleName":"","lastName":"He","suffix":""}],"badges":[],"createdAt":"2025-01-11 04:58:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5807293/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5807293/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13023-025-04075-7","type":"published","date":"2025-12-23T15:57:48+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":77356434,"identity":"08c64828-ef3a-4781-97a9-1f0653948da9","added_by":"auto","created_at":"2025-02-27 18:07:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1192944,"visible":true,"origin":"","legend":"\u003cp\u003ePedigrees of 18 families (20 patients) with CDG. Squares and circles represent males and females, respectively, while diamonds indicate cases of prenatal diagnosis. A slash through a symbol denotes deceased individuals.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5807293/v1/0a7005660a0bc49f0b935838.png"},{"id":77356430,"identity":"c6222e0f-3705-4eaa-9e90-30244b75cefc","added_by":"auto","created_at":"2025-02-27 18:07:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":951253,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Proportion of individuals presenting with specific symptoms at diagnosis in our cohort.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5807293/v1/9d3463d3fb597d5a3e9aa789.png"},{"id":77356431,"identity":"3e08203f-65e9-41b1-9cae-922e1d928628","added_by":"auto","created_at":"2025-02-27 18:07:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4524307,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Distribution of \u003cem\u003eALG2 \u003c/em\u003evariants and genotype-phenotype analysis. Variants associated with CDG are highlighted in red, while those linked to CMS are marked in black. IM: intramembrane. (B) Predicted effects of \u003cem\u003eALG2\u003c/em\u003e variants identified in this study on protein stability using a three-dimensional structural model. WT: wild type.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5807293/v1/a3af563d887acb42c99cadf4.png"},{"id":77356451,"identity":"66c28209-3c51-4a83-adb8-9859774d1b0e","added_by":"auto","created_at":"2025-02-27 18:07:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1235137,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Distribution of \u003cem\u003eDPM2\u003c/em\u003e variants and genotype-phenotype analysis. Patients with variants highlighted in red exhibited milder symptoms, while those with variants in black experienced severe symptoms and died before age 3 years. (B) Distribution of \u003cem\u003eSSR4\u003c/em\u003e variants, including fragment deletions. TM: transmembrane.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5807293/v1/4b6a9071bd6b01c20a72e221.png"},{"id":99172615,"identity":"28eb319e-f8d5-4433-8cca-68b5ff683945","added_by":"auto","created_at":"2025-12-29 16:11:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10705492,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5807293/v1/6623bf1b-a253-4b1f-a5df-89a4898747fd.pdf"},{"id":77356429,"identity":"fd4f13b0-0e8d-4955-8e32-52438b1c034b","added_by":"auto","created_at":"2025-02-27 18:07:20","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":114688,"visible":true,"origin":"","legend":"","description":"","filename":"Tab.doc","url":"https://assets-eu.researchsquare.com/files/rs-5807293/v1/a4095c5d7dcd1939a002645c.doc"}],"financialInterests":"","formattedTitle":"Clinical and genetic characterization of Congenital disorders of glycosylation in 20 Chinese patients: novel variants and genotype-phenotype correlations","fulltext":[{"header":"Background","content":"\u003cp\u003eCongenital disorders of glycosylation (CDG), are a heterogeneous group of inherited metabolic disorders caused by defects in glycosylation pathways (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Glycosylation is the predominant form of post-translational modification for proteins and lipids to by transferreing specific amino acid residues by glycosyltransferases, and plays an important role in protein folding, immune recognition, and other physiological processes(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Glycosylation is ubiquitous, occurring in every cell and organism, with over half of all cellular proteins undergoing glycosylated. Consequently, CDG often present highly diverse clinical presentations and involve multisystems(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Typical clinical features of CDG include neurological and developmental disabilities, such as psychomotor retardation/cognitive impairment, epilepsy, hytotonia, and ataxia. Other systems or organs, including hepatic, gastrointestinal, hormonal, and immune systems as well as the heart, eyes, and skeleton, are usually affected. In addition, the severity of symptoms ranges widely, from perinatal death or miscarriage to mild adult-onset forms(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSince the first CDG, PMM2-CDG, was discovered in 1980, the field has been expanding rapidly\u003c/p\u003e \u003cp\u003ewith the application of next-generation sequencing in clinical practice, with over 160 subtypes identified, which enhances our understanding of these disorders. In this study, we analyzed 20 patients from China, with all probands evaluated using whole-exome sequencing (WES). Among the cohort, we identified 28 variants in several genes: \u003cem\u003eALG2\u003c/em\u003e (3 cases), \u003cem\u003eDPM2\u003c/em\u003e (3 cases), \u003cem\u003ePMM2\u003c/em\u003e (3 cases) and \u003cem\u003eALG13\u003c/em\u003e (2 cases), with the remaining patients carrying variants in \u003cem\u003eCOG5\u003c/em\u003e, \u003cem\u003eCOG6\u003c/em\u003e, \u003cem\u003eMOGS\u003c/em\u003e, \u003cem\u003eDPM3\u003c/em\u003e, \u003cem\u003eALG1\u003c/em\u003e, \u003cem\u003eALG3\u003c/em\u003e, \u003cem\u003eALG11\u003c/em\u003e, \u003cem\u003eSSR4\u003c/em\u003e and \u003cem\u003eSLC35A2\u003c/em\u003e, 11 of which are novel and have not been reported. These findings provide valuable insights into CDG genetic diversity and offer foundational clues for future diagnosis and therapeutic advancements.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eStudy subjects\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePatients with initial symptoms such as seizures, growth retardation, delayed cognitive and motor development, and visual or speech impairments were investigated. Patients with symptoms attributed to environmental and non-genetic factors were excluded. Detailed medical and clinical data, including family history, MRI neuroimaging, electroencephalography (EEG), and other relevant examinations, were collected. This study included 20 affected individuals suspicious or diagnosed of having CDG, along with their healthy family members. All patients were born into families with healthy parents and exhibited normal at birth. Six families underwent prenatal diagnosis following a confirmed diagnosis. Six families underwent prenatal diagnosis following a confirmed CDG diagnosis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe study was approved by the ethics committee of the Institutional Review Board (IRB) of Wuhan Children\u0026apos;s Hospital, Tongji Medical College, Huazhong University of Science \u0026amp; Technology, with the approval code 2021R060-E1. Written informed consent was secured from all participants whose data are presented individually in accordance with the principles outlined in the Declaration of Helsinki.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhole-exome sequencing and data analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole WES and subsequent data analysis were conducted with the help of the third party medical laboratory (Chigene Lab, Beijing China). For WES, the target DNA fragments were enriched by hybridization to construct an exome library (IDT The xGen Exome Research Panel v1.0). High-throughput sequencing was performed using an Illumina NovaSeq 6000 sequencer. Single-nucleotide and indel variants were identified using the Genome Analysis Toolkit (GATK). Paired-end alignment was performed using the Burrows-Wheeler Aligner (BWA). Moreover, ANNOVAR software annotated and filtered the variants. SNPs and indels were filtered and screened according to sequence depth and mutation quality. The variants were annotated using the OMIM, ClinVar and HGMD databases. Candidate variants were confirmed by Sanger sequencing using self-designed primers in the patients and their parents.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo predict the pathogenicity of the variant, we used several bioinformatics software and tools.\u003c/p\u003e\n\u003cp\u003eAt first, variants were preferentially selected for further analysis and validation with their minor allele frequency (MAF)\u0026lt;0.01 in the ExAc browser (https://exac.broadinstitute.org) and gnomAD (https://gnomad.broadinstitute.org) database\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eThen, single nucleotide variants(SNV) and short indel candidates were identified. The SIFT utilities were used to forecast the change in protein structure. Conservation of each amino acid change was calculated using PhyloP2 and MutationTaster (http://www.mutationtaster.org/) algorithms were used to predict the effects of variants on protein function. All variants were named according to the guidelines of the Human Genome Variation Society (http://www.hgvs.org/) and were described and classified based on the standard guidelines of American College of Medical Genetics and Genomics(ACMG)(8).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenotype-phenotype associations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe novel candidate variants in this study or previously reported mutations to cause CDG disease was summarized and reviewed and adapted to the clinical symptoms of each patient. They were also determined using several databases such as Human Gene Mutation Database, PubMed \u0026nbsp;(https://www.ncbi.nlm.nih.gov/pubmed/), and Online Mendelian Inheritance in Man (https://omim.org/) to analysis the association between genotype and phenotype.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eOverall characteristics and molecular diagnosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study included 20 CDG patients (13 males and 7 females) from 18 non-consanguineous families (Fig.1 and Table 1). Clinical symptoms were presented for 20 cases and cataloged using Human Phenotype Ontology (HPO) terms (Table 1). The overall median age of symptom onset was 6.0 months (range: 1.0\u0026ndash;48.0 months), and the median age at diagnosis was 1.45 years (range: 4 months to 20 years). The most common symptoms were developmental delay (100%, 19/19), followed by facial dysmorphia (64.7%, 11/17), hearing or ophthalmological problems (52.9%, 9/17), abnormal liver function (52.6%, 10/19) and seizures(50%) (Fig. 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBy using WES, we identified 28 disease-causing variants in 13 genes among the 20 patients. \u0026nbsp;Among these variants, 17 previously reported and 11 novel variants. A summary of detected mutations is provided in Table 2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe patients were followed from 6 months to 5 years in outpatient of Wuhan children\u0026rsquo;s hospatal. During this time, two patients died due to recurrent intractable epilepsy associated with their diseases (P7 and P14). Prenatal diagnosis was performed in six families (F2, F3, F7, F12, F13 and F14) using amniotic fluid cells. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatients with variants in the \u003cem\u003eALG2\u003c/em\u003e gene\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVariants in the \u003cem\u003eALG2\u003c/em\u003e gene (NM_003087) are associated with ALG2-CDG, also known as CDG Ii (OMIM#607906), which is characterized by neurological symptoms such as convulsive syndrome, epilepsy, axial hypotonia, mental and motor regression (9). Additionally, \u003cem\u003eALG2\u003c/em\u003e variants can cause congenital myasthenic syndrome type 14 (ALG2-CMS, OMIM#616228), which presents within the first decade of life, progresses slowly (10).\u003c/p\u003e\n\u003cp\u003eIn this study, three patients (P5, P6 and P7) from 2 families with compound heterozygous \u003cem\u003eALG2\u003c/em\u003e variants were included. The initial symptom of these 3 patients was epileptic seizures. All of them exhibited seizures, mental and developmental regression, severe speech defects, and abnormal liver function. Genetic analysis identified compound heterozygous variants (c.422A\u0026gt;T (p.D141V) and c.751C\u0026gt;T (p.R251C)) in P5, and variants (c.751C\u0026gt;T and c.935_937del (p.L312del)) in P6 and P7. According to standard guidelines of ACMG, c.751C\u0026gt;T was classified as LP, while c.422A\u0026gt;T and c.935_937del as VUS. The potential effects of these variants were predicted using a three-dimensional structural model, indicating Protein stability being reduced compared to the wild type (lower panel in Fig. 3). Based on typical clinical manifestations and genetic findings, these patients were diagnosed with ALG2-CDG. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatients with variants in the\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003cem\u003eDPM2\u003c/em\u003e gene\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDPM2-CDG (OMIM#615042) is an extremely rare subtype of CDG.In this study, P12 presented with seizures, followed by neurological and motor developmental delay, and elevated CK levels. Genetic analysis identified a homozygous variant c.176T\u0026gt;G in exon 4 of \u003cem\u003eDPM2\u003c/em\u003e (NM_003863.4), inherited from heterozygous parents. Two additional cases, P13 and P14, had been previously reported (11). Together with our case in this study, only six cases from three families have been identified (12-13).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatients with variants in \u003cem\u003eSSR4\u003c/em\u003e gene\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSSR4-CDG is a rare and relatively mild subtype of CDG (also known as CDG Iy), predominantly affecting males (14). Key clinical features include developmental delay, speech delay, intellectual disability, muscular hypotonia, microcephaly, skeletal abnormalities, and distinct facial features (15). In this study, we identified a hemizygous spanning exons 4 to 6 of \u003cem\u003eSSR4\u0026nbsp;\u003c/em\u003ein P18, inherited from his healthy mother, in a 16-month-old boy. He was born at 39 weeks following a pregnancy complicated by intrauterine growth retardation,.and present with global developmental delay, speech delay, cognitive impairment, hypotonia, frbrile seizures, feeding difficulty, and mild facial dysmorphisms (large ears, smooth and long philtrum, abnormal eye distance, prominent nose). A literature review revealed 11 point variants and 7 fragment deletions in \u003cem\u003eSSR4\u003c/em\u003e from 23 SSR4-CDG patients (15)(Fig.4B). High degree of phenotypic heterogeneity across individuals was found and no clear genotype-phenotype correlation has been established. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatients with variants in \u003cem\u003eSLC35A2\u003c/em\u003e gene\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSLC35A2-CDG, an X-linked dominant disorder, typically manifests during infancy. The condition is characterized by microcephaly, psychomotor development delay, epileptic seizures, and hypotonia (16). Male patients usually carry mosaic variants (17). A hemizygous variant (c.902C\u0026gt;T) in was \u003cem\u003eSLC35A2\u003c/em\u003e was identified in P20. He was a 35-month-old boy with short stature (\u0026lt;-3sd), mild language and motor developmental delay, mild facial dysmorphisms, pectus carinatum, and elevated aspartate transaminase (AST) levels. A literature review identified four male cases with hemizygous variants in \u003cem\u003eSLC35A2\u003c/em\u003e (18-20), and their clinical features included short stature, abnormal liver function, language delay, intellectual disability, mild facial abnormalities, skeletal deformities and epileptic seizures (19). In addition, male patients with mosaic \u003cem\u003eSLC35A2\u0026nbsp;\u003c/em\u003evariants were reported to exhibit more severe symptoms, including epileptic encephalopathy or drug-resistant focal epilepsy (21-22). Based on these findings, we propose that male patients with hemizygous \u003cem\u003eSLC35A2\u0026nbsp;\u003c/em\u003evariants generally present with milder phenotypes compared to those with mosaic variants.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatients with variants in the\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ePMM2\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;gene\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePMM2-CDG (CDG Ia, OMIM#212065) is the most common CDG with more than 1000 cases recorded worldwide (23-24). Three patients (P8, P9, and P10) presented with developmental delay and hypotonia. They also had abnormal liver functions, evidenced by elevated aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Brain MRI showed cerebellar atrophy and white matter demyelination. Compound heterozygous variants in \u003cem\u003ePMM2\u003c/em\u003e (NM_000303.3) were identified in all three cases. The variant c.406T\u0026gt;G (p.C136G) in P9 was first reported in this work and classified as LP (PM1, PM2, PP2, PP3) according to ACMG guidelines.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatients with variants in the\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003cem\u003eALG13\u0026nbsp;\u003c/em\u003egene\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eALG13-CDG (OMIM#300884) is a rare X-linked CDG, which affects the N-linked glycosylation pathway (25). Affected individuals typically exhibit refractory seizures, psychomotor development delay, poor or absent speech, hypotonia, facial dysmorphism, and intellectual disability (26). Epileptic spasm is a common presenting symptom of ALG13-CDG.\u003c/p\u003e\n\u003cp\u003eTwo unrelated patients (P16 and P17) with infantile spasms associated with hypsarrhythmia on EEG as the initial symptom was identified (Table 1). Both patients currently exhibit delayed language and motor development. In addition, P16 has the characteristic of recurrent infections. Brain MRI showed a right choroidalcyst and widening of bilateral lateral fissures intracranial space. A \u003cem\u003ede novo\u003c/em\u003e heterozygous variant c.320A\u0026gt;G (p.N107S) in \u003cem\u003eALG13\u003c/em\u003e (NM_001099922.3), a known hotspot pathogenic variant, was identified in P16. In P17, a hemizygous variant (c.999G\u0026gt;C, p.K333N) was found and classified as a VUS in patient 16, inherited from the patient\u0026apos;s affected mother.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatients with variants in COG5 and COG6 genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe COG (conserved oligomeric Golgi) complex is composed of 8 subunits, including lobe A (from subunit COG1 to COG4) and lobe B (from subunit COG5 to COG8)(27). Genetic defect seven subunits (all but COG3) disrupts the function of the COG complex and have been reported to be associated with different types of CDG. In our study, two patients (P1 and P2) had genetic variants in COG5 (c.1039C\u0026gt;T, c.928+3A\u0026gt;G) and COG6 (c.1843C\u0026gt;T, c.428G\u0026gt;T), respectively, an the patient P2 had been reported before (28). Their clinical phenotypes were shown in Table 1 and similar to previously reported cases. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatients with variants in ALG1 gene\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eALG1-CDG is an autosomal recessive disease with severe multiorgan involvement (OMIM 608540) caused by pathogenic variants in ALG1(29). The most common phenotypic manifestations are developmental delay, intellectual disability, failure to thrive, hypotonia, and epilepsy. P15, 1-year-boy, had compound heterozygous variants in \u003cem\u003eALG1\u003c/em\u003e gene (NM_019109, c.328C\u0026gt;A, and c.863-2A\u0026gt;G), and these two variants were classified as LP and P according to ACMG guidelines. This boy had drug-resistant epilepsy, facial dysmorphisms and abnormal liver function. and died at 14 month old. Prenatal diagnosis was performed for this family when his mother was pregnant and they got a healthy baby.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDespite global recognition, CDG is under reported in China, with limited case studies available. This is first comprehensive report from China to analyze 20 diagnosed CDG cases, highlighting clinical manifestations such as seizures, motor delays, facial dysmorphism, and growth retardation, supported by findings like abnormal MRI neuroimaging (e.g., cerebellar atrophy), elevated CK levels, and abnormal liver function. This study underscores the importance of integrating genomic tools and systematic evaluations to improve CDG diagnosis and characterization.\u003c/p\u003e \u003cp\u003eA recent large cohort study of 280 individuals with genetic data consistent with a CDG diagnosis found that (s) developmental delay was the most frequent presenting symptom (77%), followed by hypotonia (56%) and feeding difficulties (42%) (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Similarly, in our cohort, development delay was the most common initial symptom, observed in all individuals by the time of enrollment, followed by facial dysmorphism (64.7%), abnormal liver function (52.6%), and seizures (50%). The broad differential diagnosis associated with these symptoms, reflecting significant clinical heterogeneity, continues to pose a challenge for timely CDG diagnosis.\u003c/p\u003e \u003cp\u003eWe found 28 distinct variants, including 11 novel variants, in 13 different genes: \u003cem\u003eALG1\u003c/em\u003e, \u003cem\u003eALG2\u003c/em\u003e, \u003cem\u003eALG3\u003c/em\u003e, \u003cem\u003eALG11\u003c/em\u003e, \u003cem\u003eALG13\u003c/em\u003e, \u003cem\u003eCOG5\u003c/em\u003e, \u003cem\u003eCOG6\u003c/em\u003e, \u003cem\u003eMOGS\u003c/em\u003e, \u003cem\u003eDPM2\u003c/em\u003e, \u003cem\u003eDPM3\u003c/em\u003e, \u003cem\u003eSSR4\u003c/em\u003e, \u003cem\u003eSLC35A2\u003c/em\u003e, and \u003cem\u003ePMM2\u003c/em\u003e. In line with previously findings (38), the majority of variants were missense (78.5%), followed by nonsense (10.7%), splice site (7.1%), and frameshift deletions (3.6%) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). According to ACMG classification guidelines, 6 variants were classified as pathogenic (P), 8 as likely pathogenic (LP), and 14 as variants of uncertain significance (VUS). Autosomal recessive inheritance was observed in 80% (16/20) of cases and 20% (4/20) displayed X-linked inheritance.\u003c/p\u003e \u003cp\u003eThe most common variants were in genes associated with disorders of N-linked protein glycosylation, including \u003cem\u003ePMM2\u003c/em\u003e (3 patients from 3 families), \u003cem\u003eALG2\u003c/em\u003e(3 from 2 families), \u003cem\u003eALG13\u003c/em\u003e (2 patients), \u003cem\u003eALG1\u003c/em\u003e(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), \u003cem\u003eALG3\u003c/em\u003e(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), \u003cem\u003eALG11\u003c/em\u003e(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), \u003cem\u003eMOGS\u003c/em\u003e(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), and \u003cem\u003eSSR4\u003c/em\u003e(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), followed by genes causing disorders of dolichol metabolism (DPM2 and DPM3) and disorders of Golgi trafficking and transport (\u003cem\u003eCOG5\u003c/em\u003e, \u003cem\u003eCOG6\u003c/em\u003e, and \u003cem\u003eSLC35A2\u003c/em\u003e), which is consistent with previous report (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). This study significantly expands the current mutational spectrum of CDG, and also revealed considerable genetic heterogeneity, reflecting the complex molecular underpinnings of CDG.\u003c/p\u003e \u003cp\u003eIn this study, we conducted a correlation analysis between genotype and phenotype by retrieving all previously published cases, including our patients, for these genes associated with uncommon CDGs.\u003c/p\u003e \u003cp\u003e \u003cem\u003eALG2\u003c/em\u003e variants could develop ALG2-CDG or ALG2-CMS, by reviewing literature, a total of 9 cases of ALG2-CMS and 5 cases of ALG2-CDG were reported from 7 articles (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR32 CR33\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Together with our study, 11 variants in \u003cem\u003eALG2\u003c/em\u003e have been identified. we noticed that variants located before the intramembrane region (N-terminal, before 158aa) are associated with ALG2-CMS, while variants occurring after the intramembrane region are linked to ALG2-CDG, which presents with more severe clinical symptoms (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). ALG2 takes part in the fourth and fifth step of lipid-linked oligosaccharide (LLO) synthesis, with two different mannosyltransferase activities. Specifically, Alg2 adds both α 1,3-and α1,6-mannose onto ManGlcNAc\u003csub\u003e2\u003c/sub\u003e\u0026ndash;Pdol to form the trimannosyl core Man\u003csub\u003e3\u003c/sub\u003eGlcNAc\u003csub\u003e2\u003c/sub\u003e-PDol (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). The Alg2 conserved C-terminal EX7E motif, the N-terminal cytosolic tail, and 3G-rich loop motifs, are essential for these enzymatic activities, both in vitro and in vivo (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Variants in these regions impair the enzyme\u0026rsquo;s function, which likely contributes to the more severe clinical manifestations observed in ALG2-CDG compared to the relatively milder phenotype of CMS caused by \u003cem\u003eALG2\u003c/em\u003e variants.\u003c/p\u003e \u003cp\u003eDPM2-CDG is an extremely rare form of CDG, and the main clinical manifestations of these patients include hypotonia, intractable epilepsy, muscle damage, elevated serum creatine kinase (CK) levels, and microcephaly (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). To date, including our cases, only 7 cases from four families have been identified (\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Our analysis revealed variants affecting the first transmembrane region or disrupting it were associated with more severe symptoms, and three patients with such variants died before the age of 3 years. In contrast, variants in the second transmembrane region resulted in less severe manifestations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). DPM2 contains two putative transmembrane domains (amino acid residues 11\u0026ndash;31 and 49\u0026ndash;69) (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Variants in the first domain, such as Phe21 and Tyr23 substitutions, impair its ability to bind with DPM1 (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e), suggesting that the first transmembrane region is crucial for maintaining the stability of the complex.\u003c/p\u003e \u003cp\u003eSSR4-CDG is ultra-rare X linked, comparably mild subtype of CDG, presenting mostly in males. We reported a 16-month-old boy, who had most of the key symptoms of SSR4-CDG, developmental delay, speech delay, cognitive impairment, feeding difficulties, and muscular hypotonia. In addition, this patient had febrile seizure and ocular abnormality. However, he had no obvious microcephaly, and his facial feature is not as typical as described previously, which could be due to the young age.\u003c/p\u003e \u003cp\u003e \u003cem\u003eSLC35A2\u003c/em\u003e, located on chromosome Xp11.23, encodes UGT-1 and is part of the SLC35 family of nucleotide sugar transporters (NSTs). \u003cem\u003eDe novo\u003c/em\u003e variants in this gene are associated with SLC35A2-CDG, often with epileptic encephalopathy as a prominent feature (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Somatic \u003cem\u003eSLC35A2\u003c/em\u003e variants have also been reported in patients with focal seizures and suspected focal cortical dysplasia (FCD) (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Furthermore, recent studies have linked brain somatic variations in \u003cem\u003eSLC35A2\u003c/em\u003e to mild malformation of cortical development with oligo dendroglial hyperplasia in epilepsy (MOGHE) (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). While SLC35A2-CDG typically shows a strong gender bias, with most cases occurring in females, there have been reports of male patients with a milder phenotype, including minor neurological involvement and growth deficiency (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). By reviewing literature, we postulate that males with hemizygous variants in \u003cem\u003eSLC35A2\u003c/em\u003e may present with a milder phenotype compared to those with mosaic variants in brain, although this requires further research.\u003c/p\u003e \u003cp\u003eOur study has some limitations. First, due to the small number of patients enrolled, we were unable to analyze more specific genotype-phenotype correlations. The rarity of CDG and the limited screening of this condition in China may partly explain this limitation. Second, changes in glycan composition in patient serum were not analyzed by MALDI-TOF MS due to the lack of necessary equipment. In addition, we identified some variants of VUS, functional studies were not conducted. Further research with larger patient cohorts and long-term follow-up would enhance our understanding of these diseases, which is critical for elucidating pathogenic mechanisms, improving the genotype-phenotype correlation, refining genetic counseling, and developing new treatments.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe identified 28 disease-causing variants, including 11 novel variants, using WES in 20 CDG patients. Our findings expand the spectrum of known variants and their related clinical phenotypes in Chinese CDG patients and establish possible genotype-phenotype correlations of several genes.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCDG: Congenital disorders of glycosylation\u003c/p\u003e\n\u003cp\u003eVUS: variants of uncertain significance\u003c/p\u003e\n\u003cp\u003eNST: nucleotide sugar transporters\u003c/p\u003e\n\u003cp\u003eWES: whole-exome sequencing\u003c/p\u003e\n\u003cp\u003eEEG:electroencephalography\u003c/p\u003e\n\u003cp\u003eGATK: Genome Analysis Toolkit\u003c/p\u003e\n\u003cp\u003eBWA: Burrows-Wheeler Aligner\u003c/p\u003e\n\u003cp\u003eHPO: Human Phenotype Ontology\u003c/p\u003e\n\u003cp\u003eFCD: focal cortical dysplasia\u003c/p\u003e\n\u003cp\u003eLLO: lipid-linked oligosaccharide\u003c/p\u003e\n\u003cp\u003eAST: aspartate aminotransferase\u003c/p\u003e\n\u003cp\u003eALT: alanine aminotransferase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSNV: single nucleotide variants\u003c/p\u003e\n\u003cp\u003eMAF: minor allele frequency\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cstrong\u003e: t\u003c/strong\u003ehe study was approved by the ethics committee of the Institutional Review Board (IRB) of Wuhan Children\u0026apos;s Hospital, Tongji Medical College, Huazhong University of Science \u0026amp; Technology, with the approval code 2020R006-E05. Written informed consent was secured from all participants whose data are presented individually in accordance with the principles outlined in the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication：\u003c/strong\u003ethe consent for publication was obtained from our patients or their guardian.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003eall data and materials are available from the corresponding on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e: the authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eThis work was supported by the grants of Hubei Provincial Natural Science Foundation Project (No.2023AFB893); Construction Project of Clinical Medical Research Center for Neurodevelopmental Disorders in Children in Hubei Province (HST2020-19); and Construction Project of Clinical Medical Research Center for Birth Defect Prevention and Treatment in Wuhan (WK[2023]123-4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy concepts: Xuelian He, Shiqiong Zhou, Peiwei Zhao\u003c/p\u003e\n\u003cp\u003eStudy design: Peiwei Zhao, Li Tan\u003c/p\u003e\n\u003cp\u003eLiterature research: Qingjie Meng, Peiwei Zhao, Yanqiu Hu\u003c/p\u003e\n\u003cp\u003eClinical information collection: Shiqiong Zhou, Qingjie Meng, Yanqiu Hu\u003c/p\u003e\n\u003cp\u003eData acquisition: Qingjie Meng, Xiankai Zhang, Lei Zhang, Yanqiu Hu\u003c/p\u003e\n\u003cp\u003eData analysis/interpretation: Lei Zhang, Qingjie Meng, Xiankai Zhang, Li Tan\u003c/p\u003e\n\u003cp\u003eManuscript preparation: Xuelian He, Peiwei Zhao\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eManuscript editing: Xuelian He, Shiqiong Zhou\u003c/p\u003e\n\u003cp\u003eManuscript final version approval: Xuelinan He, Shiqiong Zhou\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement:\u003c/strong\u003e we appreciate the patient participating in this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eFrancisco R, Brasil S, Poejo J, et al. Congenital disorders of glycosylation (CDG): state of the art in 2022. Orphanet J Rare Dis. 2023;18(1):329. Published 2023 Oct 19. doi:10.1186/s13023-023-02879-z\u003c/li\u003e\n \u003cli\u003ePaprocka J, Jezela-Stanek A, Tylki-Szymańska A, Grunewald S. Congenital Disorders of Glycosylation from a Neurological Perspective. Brain Sci. 2021;11(1):88. doi:10.3390/brainsci11010088\u003c/li\u003e\n \u003cli\u003eFreeze HH. Genetic defects in the human glycome. Nat Rev Genet. 2006;7(7):537-551. doi:10.1038/nrg1894\u003c/li\u003e\n \u003cli\u003eAbu Bakar N, Lefeber DJ, van Scherpenzeel M. Clinical glycomics for the diagnosis of congenital disorders of glycosylation. J Inherit Metab Dis. 2018;41(3):499-513. doi:10.1007/s10545-018-0144-9\u003c/li\u003e\n \u003cli\u003eMarques-da-Silva D, Dos Reis Ferreira V, Monticelli M, et al. Liver involvement in congenital disorders of glycosylation (CDG). A systematic review of the literature. J Inherit Metab Dis. 2017;40(2):195-207. doi:10.1007/s10545-016-0012-4\u003c/li\u003e\n \u003cli\u003eNg BG, Freeze HH. Perspectives on Glycosylation and Its Congenital Disorders. Trends Genet. 2018;34(6):466-476. doi:10.1016/j.tig.2018.03.002\u003c/li\u003e\n \u003cli\u003eAltassan R, P\u0026eacute;anne R, Jaeken J, et al. International clinical guidelines for the management of phosphomannomutase 2-congenital disorders of glycosylation: Diagnosis, treatment and follow up. J Inherit Metab Dis. 2019 May;42(3):577. doi: 10.1002/jimd.12082\u003c/li\u003e\n \u003cli\u003eRichards S, Aziz N, Bale S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17(5):405-424. doi:10.1038/gim.2015.30\u003c/li\u003e\n \u003cli\u003eThiel C, Schwarz M, Peng J, et al. A new type of congenital disorders of glycosylation (CDG-Ii) provides new insights into the early steps of dolichol-linked oligosaccharide biosynthesis. \u003cem\u003eJ Biol Chem\u003c/em\u003e. 2003;278(25):22498-22505. doi:10.1074/jbc.M302850200\u003c/li\u003e\n \u003cli\u003eCossins J, Belaya K, Hicks D, et al. Congenital myasthenic syndromes due to mutations in ALG2 and ALG14. \u003cem\u003eBrain\u003c/em\u003e. 2013;136(Pt 3):944-956. doi:10.1093/brain/awt010\u003c/li\u003e\n \u003cli\u003eZhao P, Hu Y, Hu J, et al. Identification and characterization of a new variation in DPM2 gene in two Chinese siblings with mild intellectual impairment. Front Genet. 2023;14:930692. doi:10.3389/fgene.2023.930692\u003c/li\u003e\n \u003cli\u003eBarone R, Aiello C, Race V, et al. DPM2-CDG: a muscular dystrophy-dystroglycanopathy syndrome with severe epilepsy. Ann Neurol. 2012;72(4):550-558. doi:10.1002/ana.23632\u003c/li\u003e\n \u003cli\u003eRadenkovic S, Fitzpatrick-Schmidt T, Byeon SK, et al. Expanding the clinical and metabolic phenotype of DPM2 deficient congenital disorders of glycosylation. Mol Genet Metab. 2021;132(1):27-37. doi:10.1016/j.ymgme.2020.10.007\u003c/li\u003e\n \u003cli\u003eNg BG, Raymond K, Kircher M, et al. Expanding the Molecular and Clinical Phenotype of SSR4-CDG. Hum Mutat. 2015;36(11):1048-1051. doi:10.1002/humu.22856\u003c/li\u003e\n \u003cli\u003eJohnsen C, Tabatadze N, Radenkovic S, et al. SSR4-CDG, an ultra-rare X-linked congenital disorder of glycosylation affecting the TRAP complex: Review of 22 affected individuals including the first adult patient. Mol Genet Metab. 2024;142(3):108477. doi:10.1016/j.ymgme.2024.108477\u003c/li\u003e\n \u003cli\u003eNg BG, Buckingham KJ, Raymond K, et al. Mosaicism of the UDP-galactose transporter SLC35A2 causes a congenital disorder of glycosylation. Am J Hum Genet. 2013;92(4):632-636. doi:10.1016/j.ajhg.2013.03.012\u003c/li\u003e\n \u003cli\u003eElziny S, Crino PB, Winawer M. SLC35A2 somatic variants in drug resistant epilepsy: FCD and MOGHE. Neurobiol Dis. 2023;187:106299. doi:10.1016/j.nbd.2023.106299\u003c/li\u003e\n \u003cli\u003eVals MA, Ashikov A, Ilves P, et al. Clinical, neuroradiological, and biochemical features of SLC35A2-CDG patients. J Inherit Metab Dis. 2019;42(3):553-564. doi:10.1002/jimd.12055\u003c/li\u003e\n \u003cli\u003eQuelhas D, Correia J, Jaeken J, et al. SLC35A2-CDG: Novel variant and review. Mol Genet Metab Rep. 2021;26:100717. Published 2021 Jan 30. doi:10.1016/j.ymgmr.2021.100717\u003c/li\u003e\n \u003cli\u003eKodr\u0026iacute;kov\u0026aacute; R, Pakanov\u0026aacute; Z, Krchň\u0026aacute;k M, et al. N-Glycoprofiling of SLC35A2-CDG: Patient with a Novel Hemizygous Variant. Biomedicines. 2023;11(2):580. doi:10.3390/biomedicines11020580\u003c/li\u003e\n \u003cli\u003eBarba C, Blumcke I, Winawer MR, et al. Clinical Features, Neuropathology, and Surgical Outcome in Patients With Refractory Epilepsy and Brain Somatic Variants in the SLC35A2 Gene. Neurology. 2023;100(5):e528-e542. doi:10.1212/WNL.0000000000201471\u003c/li\u003e\n \u003cli\u003eElziny S, Crino PB, Winawer M. SLC35A2 somatic variants in drug resistant epilepsy: FCD and MOGHE. Neurobiol Dis. 2023;187:106299. doi:10.1016/j.nbd.2023.106299\u003c/li\u003e\n \u003cli\u003eMuthusamy K, Perez-Ortiz JM, Ligezka AN, et al. Neurological manifestations in PMM2-congenital disorders of glycosylation (PMM2-CDG): Insights into clinico-radiological characteristics, recommendations for follow-up, and future directions. Genet Med. 2024;26(2):101027. doi:10.1016/j.gim.2023.101027\u003c/li\u003e\n \u003cli\u003eVaes L, Rymen D, Cassiman D, et al. Genotype-Phenotype Correlations in PMM2-CDG. Genes (Basel). 2021;12(11):1658. doi:10.3390/genes12111658\u003c/li\u003e\n \u003cli\u003eShah R, Eklund EA, Radenkovic S, et al. ALG13-Congenital Disorder of Glycosylation (ALG13-CDG): Updated clinical and molecular review and clinical management guidelines. Mol Genet Metab. 2024;142(2):108472. doi:10.1016/j.ymgme.2024.108472\u003c/li\u003e\n \u003cli\u003eAlsharhan H, He M, Edmondson AC, et al. ALG13 X-linked intellectual disability: New variants, glycosylation analysis, and expanded phenotypes. J Inherit Metab Dis. 2021;44(4):1001-1012. doi:10.1002/jimd.12378\u003c/li\u003e\n \u003cli\u003eWang X, Han L, Wang XY, et al. Identification of Two Novel Mutations in COG5 Causing Congenital Disorder of Glycosylation. Front Genet. 2020;11:168. doi:10.3389/fgene.2020.00168\u003c/li\u003e\n \u003cli\u003eNg BG, Shiryaev SA, Rymen D, et al. ALG1-CDG: Clinical and Molecular Characterization of 39 Unreported Patients. Hum Mutat. 2016;37(7):653-660. doi:10.1002/humu.22983\u003c/li\u003e\n \u003cli\u003eLam C, Scaglia F, Berry GT, et al. Frontiers in congenital disorders of glycosylation consortium, a cross-sectional study report at year 5 of 280 individuals in the natural history cohort. Mol Genet Metab. 2024;142(4):108509. doi:10.1016/j.ymgme.2024.108509\u003c/li\u003e\n \u003cli\u003eK\u0026auml;mpf M, Absmanner B, Schwarz M, Lehle L. Biochemical characterization and membrane topology of Alg2 from Saccharomyces cerevisiae as a bifunctional alpha1,3- and 1,6-mannosyltransferase involved in lipid-linked oligosaccharide biosynthesis. \u003cem\u003eJ Biol Chem\u003c/em\u003e. 2009;284(18):11900-11912. doi:10.1074/jbc.M806416200\u003c/li\u003e\n \u003cli\u003eMart\u0026iacute;nez Duncker I, Mata-Salgado D, Shammas I, et al. Case report: Novel genotype of ALG2-CDG and confirmation of the heptasaccharide glycan (NeuAc-Gal-GlcNAc-Man2-GlcNAc2) as a specific diagnostic biomarker. \u003cem\u003eFront Genet\u003c/em\u003e. 2024;15:1363558. doi:10.3389/fgene.2024.1363558\u003c/li\u003e\n \u003cli\u003eMonies DM, Al-Hindi HN, Al-Muhaizea MA, et al. Clinical and pathological heterogeneity of a congenital disorder of glycosylation manifesting as a myasthenic/myopathic syndrome. \u003cem\u003eNeuromuscul Disord\u003c/em\u003e. 2014;24(4):353-359. doi:10.1016/j.nmd.2013.12.010\u003c/li\u003e\n \u003cli\u003ePapazoglu GM, Cubilla M, Pereyra M, et al. Mass spectrometry glycophenotype characterization of ALG2-CDG in Argentinean patients with a new genetic variant in homozygosis. \u003cem\u003eGlycoconj J\u003c/em\u003e. 2021;38(2):191-200. doi:10.1007/s10719-021-09976-w\u003c/li\u003e\n \u003cli\u003eEhrstedt C, Liu WW, Frykholm C, Beeson D, Punga AR. Novel pathogenic ALG2 mutation causing congenital myasthenic syndrome: A case report. \u003cem\u003eNeuromuscul Disord\u003c/em\u003e. 2022;32(1):80-83. doi:10.1016/j.nmd.2021.11.012\u003c/li\u003e\n \u003cli\u003eLi ST, Wang N, Xu XX, et al. Alternative routes for synthesis of N-linked glycans by Alg2 mannosyltransferase. \u003cem\u003eFASEB J\u003c/em\u003e. 2018;32(5):2492-2506. doi:10.1096/fj.201701267R\u003c/li\u003e\n \u003cli\u003eMaeda Y, Tomita S, Watanabe R, Ohishi K, Kinoshita T. DPM2 regulates biosynthesis of dolichol phosphate-mannose in mammalian cells: correct subcellular localization and stabilization of DPM1, and binding of dolichol phosphate. \u003cem\u003eEMBO J\u003c/em\u003e. 1998;17(17):4920-4929. doi:10.1093/emboj/17.17.4920\u003c/li\u003e\n \u003cli\u003eBonduelle T, Hartlieb T, Baldassari S, et al. Frequent SLC35A2 brain mosaicism in mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (MOGHE). \u003cem\u003eActa Neuropathol Commun\u003c/em\u003e. 2021;9(1):3. Published 2021 Jan 6. doi:10.1186/s40478-020-01085-3\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"orphanet-journal-of-rare-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ojrd","sideBox":"Learn more about [Orphanet Journal of Rare Diseases](http://ojrd.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ojrd/default.aspx","title":"Orphanet Journal of Rare Diseases","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Congenital disorders of glycosylation, genetic variants, genotype-phenotype correlation, whole-exome sequencing, novel variants","lastPublishedDoi":"10.21203/rs.3.rs-5807293/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5807293/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eCongenital disorders of glycosylation (CDG) are a complex and heterogeneous family of rare metabolic diseases that affect protein and lipid glycosylation and glycosylphosphatidylinositol synthesis. These disorders can affect multiple organs, leading to a broad spectrum of symptoms that vary among different CDG subtypes and between individuals with same type of CDG. This study aimed to investigate the genetic variants, molecular etiologies, and clinical features of 20 Chinese patients diagnosed with CDG.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eUsing whole-exome sequencing (WES), functional prediction tools, Sanger sequencing, and segregation analysis, we identified variants in several genes: \u003cem\u003eALG2\u003c/em\u003e (3 patients), \u003cem\u003eDPM2\u003c/em\u003e (3 patients), \u003cem\u003ePMM2\u003c/em\u003e(3 patients), and \u003cem\u003eALG13\u003c/em\u003e (2 patients). Additionally, variants in \u003cem\u003eCOG5\u003c/em\u003e, \u003cem\u003eCOG6\u003c/em\u003e, \u003cem\u003eMOGS\u003c/em\u003e, \u003cem\u003eDPM3\u003c/em\u003e, \u003cem\u003eALG1\u003c/em\u003e, \u003cem\u003eALG3\u003c/em\u003e, \u003cem\u003eALG11\u003c/em\u003e, \u003cem\u003eSSR4\u003c/em\u003e and \u003cem\u003eSLC35A2\u003c/em\u003e each were observed in single case. In total, 28 distinct variants were identified, 11 of which were previously unreported. Genotype-phenotype correlations revealed notable findings: variants in the N-terminus of \u003cem\u003eALG2\u003c/em\u003e before the intramembrane domain were associated with congenital myasthenic syndromes (CMS), whereas those in the C-terminus caused ALG2-CDG; DPM2-CDG patients with variants in transmembrane region 1 exhibited more severe phenotypes; male patients with hemizygous variants in \u003cem\u003eSLC35A2\u003c/em\u003e demonstratedmilder phenotypes compared to those with mosaic variants.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThis findings expand the spectrum of known clinical presentations and genetic variants in CDG, and establish possible genotype-phenotype correlations of several pathogenic genes, emphasizing the need for functional studies to unravel the underlying mechanisms.\u003c/p\u003e","manuscriptTitle":"Clinical and genetic characterization of Congenital disorders of glycosylation in 20 Chinese patients: novel variants and genotype-phenotype correlations","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-27 18:07:14","doi":"10.21203/rs.3.rs-5807293/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2025-04-24T14:58:33+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-02-27T21:36:57+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-02-24T17:13:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-13T05:48:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Orphanet Journal of Rare Diseases","date":"2025-01-10T23:57:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"orphanet-journal-of-rare-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ojrd","sideBox":"Learn more about [Orphanet Journal of Rare Diseases](http://ojrd.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ojrd/default.aspx","title":"Orphanet Journal of Rare Diseases","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8cd0e74b-9122-4aac-811a-408e75b8a966","owner":[],"postedDate":"February 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-29T16:07:32+00:00","versionOfRecord":{"articleIdentity":"rs-5807293","link":"https://doi.org/10.1186/s13023-025-04075-7","journal":{"identity":"orphanet-journal-of-rare-diseases","isVorOnly":false,"title":"Orphanet Journal of Rare Diseases"},"publishedOn":"2025-12-23 15:57:48","publishedOnDateReadable":"December 23rd, 2025"},"versionCreatedAt":"2025-02-27 18:07:14","video":"","vorDoi":"10.1186/s13023-025-04075-7","vorDoiUrl":"https://doi.org/10.1186/s13023-025-04075-7","workflowStages":[]},"version":"v1","identity":"rs-5807293","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5807293","identity":"rs-5807293","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}