ALG6-CDG, formerly known as CDG-Ic, is a rare inherited condition that affects many systems in the body. It is one of the more frequent congenital disorders of glycosylation (CDG) with more than 89 patients reported in the medical literature to date. ALG6-CDG is classified as a disorder of N-linked protein glycosylation. ALG6-CDG is caused when an individual has mutations in both copies of their ALG6 gene, which provides instructions for making an enzyme that attaches the sugar glucose to sugar chains during N-glycosylation. Mutations in the ALG6 gene cause proteins to have incomplete or absent sugar chains. Symptoms of ALG6-CDG begin at infancy and are primarily characterized by neurological abnormalities, including developmental delay, weakness in muscles close to the core of the body, low muscle tone, epilepsy, and problems with coordination and balance. Feeding difficulties and gastrointestinal problems are also common. Some individuals may have limb abnormalities and/or show behavioral problems. Several screening tests are available for ALG6-CDG, but a definitive diagnosis is achieved through genetic testing. There are currently no approved treatments for ALG6-CDG. Treatment is focused on the management of specific symptoms and preventing complications.
Alpha-1,3-glucosyltransferase congenital disorder of glycosylation (ALG6-CDG) is a rare autosomal recessive genetic disorder1. The first reported case of ALG6-CDG was in 19982–4 and there are 89 confirmed cases to date2–12. The ALG6 (asparagine-linked glycosylation 6) gene encodes a glucosyltransferase enzyme responsible for adding the first glucose residue during synthesis of the lipid-linked oligosaccharide (LLO) in the endoplasmic reticulum (ER)1. LLO synthesis is a precursor step to N-glycosylation, which is the process by which sugar chains (glycans) are added to the amino acid asparagine in proteins. Deficiency in the ALG6 enzyme results in the incomplete assembly of the LLO, leading to insufficient N-glycosylation of glycoproteins1.
Symptoms begin at infancy and the characteristic presentations of ALG6-CDG include neurological symptoms, gastrointestinal and feeding problems, ophthalmological problems, and behavioural abnormalities. A diagnosis can be determined through transferrin analysis and LLO analysis, but definitive diagnosis can only be achieved through genetic testing. There are currently no approved treatments for ALG6-CDG11.
- CDG syndrome type 1c
- Congenital disorder of glycosylation type 1c
- Glucosyltransferase 1 deficiency
- Alpha-1,3-glucosyltransferase congenital disorders of glycosylation
ALG6-CDG is an autosomal recessive disorder, meaning an affected individual inherits one defective copy of the gene from each asymptomatic parent11.
The ALG6 gene encodes a glucosyltransferase enzyme, ALG6 (alpha-1,3-glucosyltransferase). Glucosyltransferases are enzymes that enable the transfer of glucose molecules during glycosylation. ALG6 is located in the ER membrane where it has a role in the assembly of the LLO—the oligosaccharide precursor in protein N-glycosylation11. The ALG6 enzyme transfers the first glucose from dolichol-phosphate-glucose (Dol-P-glucose) to the growing oligosaccharide chain of the LLO prior to its attachment to a protein.
N-glycosylation is the process by which an oligosaccharide is attached to the nitrogen atom of an asparagine residue on a protein. N-glycosylation is initiated in the ER and begins with the synthesis of the LLO13. The LLO is comprised of a 14-sugar oligosaccharide, which is sometimes referred to as the N-glycan precursor oligosaccharide, attached to the lipid carrier dolichol pyrophosphate (Dol-PP). The 14-sugar glycan chain is made up of 2 N-acetylglucosamine (GlcNAc), 9 mannose, and 3 glucose residues. Once assembled, the oligosaccharide is transferred “en bloc” to proteins and undergoes further processing in the ER and Golgi13. Once the oligosaccharide is attached to a protein, it is referred to as an N-glycan.
LLO synthesis is carried out by a series of enzymes encoded by the DPAGT1 and ALG genes and can be divided into two phases: Phase I and Phase II (Figure 1)13.
Phase I of LLO synthesis takes place on the cytoplasmic side of the ER. It begins with the sequential attachment of two N-acetylglucosamine (GlcNAc2) and five mannose (Man5) residues to Dol-PP by several ER enzymes. GlcNAc and mannose are transferred from nucleotide sugars UDP-acetylglucosamine and GDP-mannose, respectively. The intermediate structure, Dol-PP-GlcNAc2Man5, is translocated across the ER membrane into the lumen the RFT1 enzyme1,13.
Phase II of LLO synthesis takes place in the ER lumen. Once in the lumen, four mannose residues followed by three glucose (Glc3) residues are added to the intermediate structure, generating the complete LLO, Dol-PP-GlcNAc2Man9Glc3. Mannose and glucose are transferred from glycosyl donors Dol-P-mannose and Dol-P-glucose, which are formed on the cytoplasmic side of the ER and must also be flipped across the ER membrane13. ALG6 is responsible for adding the first glucose residue to the growing oligosaccharide1.
Once assembled, the oligosaccharide is transferred “en bloc” from Dol-PP to asparagine residues of newly synthesized proteins via the enzyme oligosaccharyltransferase (OST), resulting in N-glycosylation of the protein13. The activity of OST is highly specific for the completely assembled 14-sugar oligosaccharide, Glc3Man9GlcNAc2. The glucose residue added to the oligosaccharide by ALG6 is also important for proper folding and proofreading of the glycoprotein in the ER and Golgi.
Mutations in the ALG6 gene lead to the production of an abnormal enzyme with reduced or no activity. In the absence of ALG6, the LLO is missing three glucose residues and is therefore incompletely assembled. This results in reduced transfer efficiency of the oligosaccharide to the protein by OST, resulting in N-glycoproteins that are insufficiently glycosylated (hypoglycosylation)1,11.
The ALG6 gene is found on chromosome 1 (1p31.1), and 23 mutations have been reported for the ALG6 gene including 11 missense mutations, 1 nonsense mutation, 5 deletion mutations, 4 splicing mutations, and 1 duplication mutation15. Missense mutations p.AV333V and p.1299Del are common mutations in ALG6, causing low muscle tone (hypotonia), speech disability, and epilepsy11,16.
Signs & Symptoms
Individuals with ALG6-CDG typically develop signs and symptoms during infancy. ALG6-CDG is primarily characterized by a mild to severe neurological disorder and feeding problems. The characteristic clinical presentations of ALG6-CDG include11:
- Neurological – global developmental delay, speech delay, motor delay, low muscle tone (hypotonia), proximal muscle weakness, lack of coordination and muscle control (ataxia), and epilepsy
- Gastrointestinal – feeding difficulties and intestinal problems may lead to a failure to gain weight and slower than normal growth (failure to thrive). Protein-losing enteropathy (PLE), one of the more life-threatening symptoms, causing an excess loss of proteins in the gastrointestinal tract17
- Ophthalmological – misaligned or crossed eyes (strabismus), repetitive and uncontrolled eye movements (nystagmus), and visual loss
- Behavioural – autistic behaviour, periodic mood swings, episodes of aggressive behaviour, and sleep disturbances
Limb anomalies appear to be a less common but a unique feature of ALG6-CDG. Other less common clinical presentations include skeletal abnormalities, such as disproportionately short fingers and toes (brachydactyly), disproportionately long fingers (arachnodactyly), limited extension of joints, abnormal facial features (facial dysmorphisms), bleeding and clotting problems, heart and liver abnormalities, abnormal fat distribution, and cerebellar abnormalities9,10,17.
Biochemical abnormalities observed in individuals with ALG6-CDG include coagulation abnormalities, low blood sugar (hypoglycemia), low serum cholesterol (hypocholesterolemia), endocrine abnormalities, increased serum transaminases, and anemia.
ALG6-CDG is classified as a disorder of N-linked protein glycosylation.
Under the former CDG classification system, ALG6-CDG is classified as a Type 1 CDG, which arise due to defects in the synthesis of oligosaccharides or their transfer to proteins.
Although diagnosis of ALG6-CDG may be suspected based on presentation of symptoms and a detailed patient history, direct molecular genetic testing is the only definitive diagnostic test. Screening in suspected patients begins with a blood test to analyze serum transferrin11. LLO analysis in patient fibroblasts may also be carried out following transferrin screening16. Diagnostic results by these tests appear to be consistent across ALG6-CDG patients11.
Individuals with ALG6-CDG show a type 1 pattern by transferrin isoelectric focusing (TIEF) or mass spectrometry analysis of transferrin8. Type 1 patterns are observed in CDG that arise due to defects in LLO assembly and are characterized by a decrease in tetrasialo-transferrin and an increase in disialo- and asialo- transferrin isoforms8,18.
LLO analysis of ALG6-CDG fibroblasts shows an increase in the intermediate LLO structure, Dol-PP-GlcNAc2Man9, which lacks three glucose residues15.
A biomarker that is unique to ALG6-CDG has not been identified.
Prognosis of ALG6-CDG may vary depending on the severity of an individual’s symptoms. Most ALG6-CDG patients are young and long-term prognosis information is unavailable. As of 2016, the oldest patient recorded in medical literature has reached their forties11. Milder ALG6-CDG cases may only present with hypotonia and seizures or behavioral abnormalities and speech disability. Severe forms of ALG6-CDG can lead to early death from complications of sepsis, seizures, or PLE. Seizures can change as patients get older, with some patients developing seizures that are resistant to therapies11.
Management of symptoms may include combinations of physical therapy, occupational therapy, speech or vision therapy, and palliative measures.
Sleep disturbances may be managed with daily melatonin treatment11. Failure to thrive patients can be put on a special diet for increased calorie intake11. PLE may be treated with a low-fat/elementary protein diet and subcutaneous treatment with octreotide11,19.
There are currently no treatment options available for ALG6-CDG. Treatment is focused on management of symptoms and prevention of complications.
Several ALG6 research models have been generated including in yeast, fly, mice, and a hamster cell line.
Yeast (S. cerevisiae)
Alg6 mutants are unable to transfer glucose from dolichol phosphoglucose in LLO synthesis, leading to an accumulation of GlcNAcMan9. Alg6 mutants have a shorter LLO glucose chain, causing growth defects and, in some cases, arrested growth20,21.
Fly (D. melanogaster)
Drosophila gene garnysstan (gny, CG5091, FBgn0032234) is orthologous to ALG6, and also encodes the alpha-1,3-glucosyltransferase enzyme. It is a potential model for the human disease ALG6-CDG (FlyBase).
Mouse (M. musculus)
Alg6 knockout mouse
Heterozygous and homozygous whole body Alg6 knockout mice have been generated from the mouse line Alg6em1(IMPC)Tcp. Heterozygous mutants live to early adulthood and phenotypically have abnormal skin morphology, enlarged urinary bladder, and enlarged lymph nodes. Homozygous mutants live to E9.5-E15.5 and phenotypically show embryonic growth retardation, abnormal embryo development, abnormal allantois morphology, abnormal embryo size, and prenatal lethality prior to hear atrial septation (MGI, IMPC).
Alg6 conditional-ready floxed knockout embryonic stem cell line
Embryonic stem (ES) cell line Alg6tm1a(EUCOMM)Hmgu is a targeted knockout/null mutation of Alg6. A “conditional-ready” allele can be created by flp recombinase expression in mice carrying this allele, and cre expression results in a knockout mouse. If cre is expressed without flp expression, a reporter knockout mouse can be generated (MGI).
Alg6 knockout ES cell line
ES cell line Alg6tm1e(EUCOMM)Hmgu is a targeted knockout/null mutation of Alg6 (MGI).
Hamster (C. griseus)
Chinese hamster ovary (CHO) cell line MI8-5 is deficient in Alg6 (Alg6–/–). The cells have low expression of the OST enzyme required for glycosylation are unable to synthesize oligosaccharides containing glucose22.
Clinical and Basic Investigations into Congenital Disorders of Glycosylation (NCT04199000)
The Frontiers in Congenital Disorder of Glycosylation Disorders Consortium (FCDGC) is conducting a 5-year natural history study on all CDG types, including ALG6-CDG. The purpose of this study is to define the natural history and clinical symptoms of CDG, develop new diagnostic techniques, identify clinical biomarkers that can be used in future clinical trials and evaluate whether dietary treatments improve clinical symptoms and quality of life.
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