ALG1-CDG: A new case with early fatal outcome

doi:10.1016/j.gene.2013.10.013

Gene

Volume 534, Issue 2, 25 January 2014, Pages 345-351

https://doi.org/10.1016/j.gene.2013.10.013 Get rights and content

Highlights

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A new patient with congenital disorder of glycosylation type Ik (ALG1-CDG).
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The boy had a severe multiorgan involvement with death in early infancy.
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Compound heterozygosity was found for c.1145 T > C (M382T) and c.1312C > T (R438W).
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Previously reported speculation on R438W as a frequent polymorphism can be disproved.
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We confirmed both of the mutations as disease-causing for ALG1-CDG (CDG-Ik).

Abstract

Congenital disorders of glycosylation (CDG) are a growing group of inherited metabolic disorders where enzymatic defects in the formation or processing of glycolipids and/or glycoproteins lead to variety of different diseases.

The deficiency of GDP-Man:GlcNAc₂-PP-dolichol mannosyltransferase, encoded by the human ortholog of ALG1 from yeast, is known as ALG1-CDG (CDG-Ik). The phenotypical, molecular and biochemical analysis of a severely affected ALG1-CDG patient is the focus of this paper. The patient's main symptoms were feeding problems and diarrhea, profound hypoproteinemia with massive ascites, muscular hypertonia, seizures refractory to treatment, recurrent episodes of apnoea, cardiac and hepatic involvement and coagulation anomalies.

Compound heterozygosity for the mutations c.1145 T > C (M382T) and c.1312C > T (R438W) was detected in the patient's ALG1-coding sequence. In contrast to a previously reported speculation on R438W we confirmed both mutations as disease-causing in ALG1-CDG.

Introduction

Congenital disorders of glycosylation (CDG) affect one of the most important co- and post-translational protein modifications. Each specific step of assembly, transfer and processing of N-linked protein glycosylation in the rough endoplasmic reticulum (RER) and the Golgi complex may be impaired as well as each step in the O-glycosylation pathway of glycoproteins or the glycolipid synthesis (Jaeken, 2010, Marquardt and Denecke, 2003).

Since the first description by Jaeken et al. (1980), many different molecular defects have been described with a broad range of disease phenotypes (Theodore and Morava, 2011).

A CDG subtype with severe multiorgan involvement is ALG1-CDG (OMIM 608540), formerly known as CDG-Ik. The human ALG1 gene (OMIM 605907), also named HMT1 (human mannosyltransferase 1), encodes a transmembrane protein called GDP-Man:GlcNAc₂-PP-dolichol mannosyltransferase (alias chitobiosyldiphosphodolichol beta-mannosyltransferase; NP_061982.3; EC number: 2.4.1.142, 13 exons, 464 amino acids, 52.5 kDa molecular weight) (Takahashi et al., 2000). The mannosyltransferase plays a central role in one of the first steps of the N-glycosylation pathway of glycoproteins at the cytosolic side of the rough endoplasmatic reticulum. GlcNAc₂-PP-dolichol is extended by the first mannose in ß1,4-linkage using GDP-mannose as a substrate donor to generate Man₁GlcNAc₂-PP-dolichol (Couto et al., 1984, Marquardt and Denecke, 2003). This step is impaired in ALG1-CDG patients.

Eighteen ALG1-CDG patients with fifteen different mutations have been described with a broad clinical spectrum from early death at the second day of life to survival beyond the age of 20. (de Koning et al., 1998, Dupré et al., 2010, Grubenmann et al., 2004, Kranz et al., 2004, Morava et al., 2012, Schwarz et al., 2004, Snow et al., 2012).

Symptoms include recurrent seizures, microcephaly, developmental delay and psychomotor retardation, muscular hypotonia, coagulation abnormalities, ascites, hepatomegaly, nephrotic syndrome, ocular manifestations, deafness and dysmorphic features.

The focus of this paper is on the phenotypical, molecular and biochemical analysis of a new patient with a severe form of ALG1-CDG. The affected boy showed several symptoms typical of ALG1-CDG but also important differences and died at the age of 3 months and 23 days.

Section snippets

Analyses of carbohydrate deficient transferrin (CDT)

To identify truncated or missing chains of serum transferrin, isoelectric focusing (IEF) as well as immunoprecipitation (IMPP) and SDS-PAGE were performed as described previously (Niehues et al., 1998).

High performance liquid chromatography (HPLC)-analysis of CDT was done as described earlier (Biffi et al., 2007).

Cell culture

A skin biopsy of the patient was taken after informed consent was obtained from the parents. Fibroblasts were grown in MEM with Earle's Salts (PAA, Cölbe, Germany) supplemented with

Case report

The patient (JdL) was the first child of healthy non-consanguineous parents of Dutch and German origin with an unremarkable family history. The course of pregnancy was uneventful up to the 33rd week when premature rupture of membranes and premature labor led to preterm delivery. The boy's postnatal physical examination was characterized by Apgar scores of 6/7/8, a weight of 2120 g (25–50th percentile), a body length of 44 cm (25th percentile) and a head circumference of 30.5 cm (25–50th

Discussion

The human ALG1 protein is an unglycosylated transmembrane protein (Takahashi et al., 2000) with a large cytosolic C-terminal domain containing the catalytically active site (Haeuptle and Hennet, 2009). The protein belongs to a group of glycosyltransferases (DPAGT1, ALG1, and ALG2) that are involved in the first steps of the assembly of dolichol-linked oligosaccharides required for N-glycosylation on the cytosolic side of the ER membrane (Haeuptle and Hennet, 2009) and catalyzes the transfer of

Conflict of interests

All authors declare no conflict of interests.

Acknowledgments

We are grateful to Dr. N. v. Deenen (Institute of Plant Biology and Biotechnology, Münster), who provided the expression vector pYEX-BX (Clontech). We thank Dr. C. Neupert and Prof. M. Aebi (Microbiology, ETH Zürich) who provided the ALG1-deficient Saccharomyces cerevisiae strain PRY56.

We also thank Martina Herting, Marianne Grüneberg, Maria Plate, Ute Mangels and Lydia Vogelpohl for expert technical assistance.

We are deeply grateful to the parents of the deceased boy.

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Glycobiology

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Cited by (27)

Chemo-enzymatic synthesis of the ALG1-CDG biomarker and evaluation of its immunogenicity
2020, Bioorganic and Medicinal Chemistry Letters
Congenital disorders of glycosylation (CDG) are a growing group diseases that result from defects in genes involved in glycan biosynthesis pathways. One tetrasaccharide, i.e., Neu5Ac-α2, 6-Gal-β1, 4-GlcNAc-β1, 4-GlcNAc, was recently reported as the biomarker of ALG1-CDG, the disease caused by ALG1 deficiency. To develop a novel diagnostic method for ALG1-CDG, chemo-enzymatic synthesis of the tetrasaccharide biomarker linked to phytanyl phosphate and the biomarker’s immune stimulation were investigated in this study. The immunization study using liposomes bearing phytanyl-linked tetrasaccharide revealed that they stimulated a moderate immune response. The induced antibody showed strong binding specificity for the ALG1-CDG biomarker, indicating its potential in medical applications.
Congenital disorders of N-linked glycosylation
2020, Rosenberg’s Molecular and Genetic Basis of Neurological and Psychiatric Disease: Volume 1
The congenital disorders of glycosylation are a family of anabolic diseases, whose hallmark manifestations relate to the hypoglycosylation of proteins and lipids. These generally involve the nervous system (intellectual disability, seizures, strokes, hearing, and vision loss), with variable expression in the eye, kidneys, liver, gastrointestinal tract, heart, endocrine organs, immune system, skin, and skeleton. Most are inherited as autosomal recessive traits, with the exception of a few X-linked conditions.
The most widely used test to screen for N-linked disorders of glycosylation is carbohydrate-deficient transferrin. It is often useful to measure other glycoconjugates, such as coagulation factors, transaminases, and creatine kinase levels, when considering the possibility of an N-linked disorder of glycosylation. Enzyme assays are available for some disorders, but, increasingly, the diagnosis is made directly by gene sequencing.
Research in N-linked disorders of glycosylation has been dominated by disease discovery in the last decade, as whole-exome sequencing has become a front line diagnostic tool. Current studies include several focused on the use of monosaccharide supplements as disease modifiers, symptomatic therapy, specifically acetazolamide for ataxia, and continuing investigation of basic glycobiology.
Chromosomal uniparental disomy 16 and fetal intrauterine growth restriction
2017, European Journal of Obstetrics and Gynecology and Reproductive Biology
Citation Excerpt :
In addition, the database shows the genes CDT1 and ALG1 located at 16q24.3 and 16p13.3 are associated with autosomal-recessive diseases (Table 3), and these genes are found in the ROH of our present case. Although the family history is normal, we cannot exclude the potential effect of homozygous mutation on CDT1 (chromatin licensing and DNA replication factor 1) and ALG1 (chitobiosyldiphosphodolichol beta-mannosyltransferase) [16,17]. The result of the UPD database search indicated matUPD16 is correlated with IUGR.
There is a well-documented association between prenatally diagnosed chromosomal uniparental disomy and poor pregnancy outcome.
In this study, we identified an intrauterine growth restricted fetus carrying a maternal UPD 16 with segmental hetero- and isodisomy using the Affymetrix CytoScan HD SNP-array and the UPDtool. We also performed FISH to exclude trisomy mosaicism of chr.16. We then explored the genetic mechanisms of how imprinted genes cause clinical abnormalities. Additionally, we reviewed the mUPD16 literature, compared the clinical phenotypes of our patient with other reported cases, and assessed the loss of autosomal-recessive genes in the regions of homozygosity.
Considering UPD mechanism of potential impact on the function of the placenta, the genetic composition of chromosome 16, and the information previous literature reports, we have reason to believe that UPD16 correlates with IUGR.
Quantitative study of yeast Alg1 beta-1, 4 mannosyltransferase activity, a key enzyme involved in protein N-glycosylation
2017, Biochimica et Biophysica Acta - General Subjects
Citation Excerpt :
ALG1-CDG patients suffer from broad multisystem defects with varying degrees of clinical severity. To date, fifty-two different human ALG1 mutations have been reported, making ALG1-CDG the third most common CDG type [9–15]. The pathogenicity of human alg1 mutations has been tested by a yeast complementation assay that measures the inability of human alg1 mutant alleles to rescue the hypoglycosylation phenotype of a yeast temperature sensitive alg1 mutant [10,12].
Asparagine (N)-linked glycosylation begins with a stepwise synthesis of the dolichol-linked oligosaccharide (DLO) precursor, Glc3Man9GlcNAc2-PP-Dol, which is catalyzed by a series of endoplasmic reticulum membrane-associated glycosyltransferases. Yeast ALG1 (asparagine-linked glycosylation 1) encodes a β-1, 4 mannosyltransferase that adds the first mannose onto GlcNAc2-PP-Dol to produce a core trisaccharide Man1GlcNAc2-PP-Dol. ALG1 is essential for yeast viability, and in humans mutations in the ALG1 cause congenital disorders of glycosylation known as ALG1-CDG. Alg1 is difficult to purify because of its low expression level and as a consequence, has not been well studied biochemically. Here we report a new method to purify recombinant Alg1 in high yield, and a mass spectral approach for accurately measuring its β-1, 4 mannosyltransferase activity.
N-terminally truncated yeast His-tagged Alg1 protein was expressed in Escherichia coli and purified by HisTrap HP affinity chromatography. In combination with LC-MS technology, we established a novel assay to accurately measure Alg1 enzyme activity. In this assay, a chemically synthesized dolichol-linked oligosaccharide analogue, phytanyl-pyrophosphoryl-α-N, N′-diacetylchitobioside (PPGn2), was used as the acceptor for the β-1, 4 mannosyl transfer reaction.
Using purified Alg1, its biochemical characteristics were investigated, including the apparent K_m and V_max values for acceptor, optimal conditions of activity, and the specificity of its nucleotide sugar donor. Furthermore, the effect of ALG1-CDG mutations on enzyme activity was also measured.
This work provides an efficient method for production of Alg1 and a new MS-based quantitative assay of its activity.
Congenital Disorders of N-linked Glycosylation
2014, Rosenberg's Molecular and Genetic Basis of Neurological and Psychiatric Disease: Fifth Edition
The congenital disorders of glycosylation (CDG) are a family of anabolic diseases with variable multisystem manifestations. Most of these disorders have multisystem manifestations, in addition to a wide variety of neurologic phenotypes. The most frequent type is PMM2-CDG (phosphomannomutase deficiency), which is associated with severe cerebellar atrophy, intellectual disability, stroke-like episodes and seizures, and progressive pigmentary retinopathy and demyelinating neuropathy in classic cases. Other phenotypes have considerable overlap, and with few exceptions, are accompanied by varying systemic manifestations that may include coagulopathies, gastrointestinal disease, lipodystrophy, skeletal deformities, cardiac, renal, and eye disease. CDG should be suspected in any patient with multisystem disease and neurologic manifestations, and can be efficiently screened for in most cases using carbohydrate deficient transferrin. Enzyme analysis is clinically available for the more common disorders, and molecular diagnosis is rapidly improving for all forms of CDG. Specific therapy is available for MPI-CDG and SLC365C1-CDG.
Dysregulated proteome and N-glycoproteome in ALG1-deficient fibroblasts
2024, Proteomics

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View full text

ALG1-CDG: A new case with early fatal outcome

Highlights

Abstract

Introduction

Section snippets

Analyses of carbohydrate deficient transferrin (CDT)

Cell culture

Case report

Discussion

Conflict of interests

Acknowledgments

Clin. Biochem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Am. J. Hum. Genet.

Am. J. Hum. Genet.

Mol. Genet. Metab.

Recurrent nonimmune hydrops fetalis associated with carbohydrate-deficient glycoprotein syndrome

J. Inherit. Metab. Dis.

An efficient transformation procedure enabling long-term storage of competent cells of various yeast genera

Yeast

Guanosine diphosphate-mannose:GlcNAc2-PP-dolichol mannosyltransferase deficiency (congenital disorders of glycosylation type Ik): five new patients and seven novel mutations

J. Med. Genet.

Physical interactions between the Alg1, Alg2, and Alg11 mannosyltransferases of the endoplasmic reticulum

Glycobiology