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| Email: | mcl@mayo.edu |
| Telephone: | 800-533-1710 |
| International: | +1 855-379-3115 |
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Expires: January 4, 2027
Gisele (Gessi) Bentz Pino, CGC
Genetic Counselor
Division of Laboratory Genetics and Genomics
Mayo Clinic, Rochester, Minnesota
Kimiyo Raymond, M.D.
Assistant Professor of Laboratory Medicine and Pathology
Division of Laboratory Genetics and Genomics
Mayo Clinic, Rochester, Minnesota
Contact us: mcleducation@mayo.edu.
Hello. My name is Gessi Pino, and I’m a genetic counselor in the Biochemical Genetics Lab at Mayo Clinic. And on behalf of Dr. Kimiyo Raymond, a clinical consultant in the laboratory and an expert in CDG, I’ll be speaking on the topic of congenital disorders of glycosylation (CDG).
I have no disclosures.
Today’s plan is to briefly discuss glycosylation and its importance in human biology, to introduce the congenital disorders of glycosylation, to highlight our laboratory testing, offer strategies to screen and diagnose CDGs, and finally offer resources for the management of patients with a CDG.
The central dogma of biology is that information flows from DNA to RNA to protein. But how does the relatively small number of genes in the human genome generate the vast biological diversity and complexity that is seen in the human population?
One reason is glycosylation. Glycosylation is the post-translational modification of proteins and lipids by the addition of glycans (sugars and sugar chains) in a complex stepwise fashion in the endoplasmic reticulum, Golgi apparatus, cytosol and sarcolemmal membrane. It is a finely tuned process conserved across eukaryotes and archae species.
The resulting glycoconjugates mediate a wide variety of metabolic processes such as the maintenance of cell or tissue structure, molecular signaling, and cell-to-cell interactions. It is one of the most important post-translational activities that proteins undergo, and it’s predicted that approximately half of all proteins are glycosylated. Glycosylation then allows them to become fully functional.
Glycans are categorized according to the nature of the linkage to the protein or lipid. And some common eukaryotic glycans include proteoglycans, glycosphingolipids, GPI (glycosylphosphatidylinositol) -anchored glycoproteins, and N-linked and O-linked glycoproteins.
Our focus is on the N-glycosylation pathway, where N-linked glycans are attached to a protein backbone via an asparagine residue on the protein. N-glycosylation is well defined and illustrates the post-translational process well.
N-glycans are partially assembled on lipid donors on the cytoplasmic face of the endoplasmic reticulum (ER) and then flipped across the membrane, where the oligosaccharide assembly is completed and the transfer to the nascent protein occurs. This oligosaccharide is then trimmed and extended by the addition of one monosaccharide at a time as the protein passes through the ER and the Golgi. These glycosylation reactions are catalyzed by glycosyltransferases. In each step along the pathway of assembly or processing, an error can occur, leading to what is termed defects in glycosylation or congenital disorders of glycosylation. The red gene names, illustrated here, indicate places along the assembly and processing line where defects have been discovered (n=~60-70).
First described in 1980 by Jaak Jaeken, CDG were initially defined as defects in N-glycosylation and classified according to glycan isoform patterns seen in transferrin protein analysis. Termed type I, type 2, and mixed. However, CDG is now applied to any individual with a defect in any glycosylation pathway, so the traditional classifications, while still valid, do not sufficiently account for all the known CDG.
Glycosylation defects include those in the activation, presentation, and transport of sugar precursors, enzymatic defects in the assembly and processing of glycans, and defects in the proteins that traffic the glycosylation machinery or maintain Golgi homeostasis.
CDG are currently named by the defective gene followed by the term CDG, for example, PMM2-CDG. Clinical features are heterogeneous, and the majority of these are autosomal recessive. Most are defects in protein glycosylation although an increasing number are defects of glycolipid or proteoglycan biosynthesis.
The diagnosis of CDG is a challenge since defects in glycosylation can affect virtually every organ system. The majority are multisystemic disorders, and most have a neurological component. In general, though, the most commonly affected organs are the brain, eyes, and skeleton. Additionally, many patients present with dysmorphic features, such as inverted nipples and abnormal fat pads. Some cardinal features include: hypotonia, developmental delay, seizures, cerebellar atrophy, cutis laxa, and strabismus. Because CDG are so heterogeneous and symptoms can be non-specific, the general recommendation is to consider CDG in any unexplained disorder, particularly one with a neurological component.
Transferrin and apolipoprotein CIII isoform analysis are the initial screening tests for CDG. Alterations in the number or structure of these forms can be captured via electrospray ionization mass spectrometry, which is a powerful technique capable of detecting both N and mucin-type core-1 O-glycosylation errors. The technique is capable of processing a high volume of samples per day with minimal sample volume. Two disadvantages are that, in rare instances, affected patients may have a normal profile, and unaffected patients may have abnormalities due to secondary causes.
Additional serum N-glycan analysis along with urine oligosaccharide analysis provides specific information on glycan or oligosaccharide abnormalities, which can be informative even in the setting of normal transferrin and Apo-CIII analysis.
The results of the initial biochemical analyses should be correlated with the clinical presentation to determine the most appropriate follow-up testing strategy which may include enzyme, molecular, and/or research-based testing.
Shown here are the isoforms detected in the mass spectrometry analysis. Various combinations of fully and partially glycosylated isoforms are reported as ratios, as we’ll see in a later slide. Alterations of ratios outside the established reference ranges are reported as abnormal, and the interpretive report includes both the quantitative results and an interpretation.
Although analysis of carbohydrate-deficient transferrin and Apo-CIII reliably detects the many N-linked hypoglycosylation disorders and mucin type core 1 O-linked disorders, it does not necessarily distinguish among those identified. This requires additional testing.
The normal state of transferrin is a fully glycosylated state represented by tetra-sialotransferrin. In a normal transferrin profile, the fully glycosylated transferrin (red circle) is abundant with very small fractions of di-sialo and a-sialo transferrin (circled in blue).
In a normal Apo-CIII profile, all three forms are present. However, the proportion becomes different in abnormal glycosylation. Usually, the most abundant is the mono-sialo,and the di-sialo will be about 50-70% of the peak height of the monosialo. In abnormal glycosylation, this ratio becomes lower.So we say the fully glycosylated Apo-CIII is low.
Also, a-sialo a-glycan Apo-CIII is usually less than 10% of the di-sialo Apo-CIII. In abnormal glycosylation, this fraction tends to become higher. So that we say the hypo glycosylated Apo-CIII is elevated.
Total N-glycan analysis by MALDI-TOF, as is performed in the CDGN assay, is a global assessment of glycosylation. Abnormal glycans are directly detected via the instrument. This complements the current CDG testing by providing an analysis that is not isolated to a single surrogate glycosylated protein, such as transferrin, and yields information on structural abnormalities that may in turn guide molecular testing.
Oligosaccharide analysis by MALDI is an assessment of excess simple carbohydrate molecules in the urine. Oligosaccharidoses, such as alpha-mannosidosis, fucosidosis, and others, are typically detected and first thought of when performing this test.However, several CDGs (MOGS-CDG, NGLY1-CDG, PMM2, and ALG1-CDGs) can also be detected with this method. This is particularly helpful for the MOGS and NGLY1-CDG since they can only be detected in the urine.
With more than two decades of experience in clinical biochemical testing for CDG, our laboratory has experience unequaled in the industry. The complexities and variations of the transferrin and Apo-CIII isoforms require skill and expertise to interpret accurately. With more than 150 described types of CDG, and new types continually being discovered, even small differences in the glycan profile can be indicative of a different type of CDG. We currently have over 300 confirmed positive patient profiles for reference, and our CLIR bioinformatics software has been utilized to establish normal transferrin and Apo-CIII reference ranges.
The case breakdown in the laboratory of those confirmed cases shows the various CDG types and number of patients per type in parentheses. Slightly less than half of all cases are PMM2-CDG, which corresponds to the literature reports of this being the most common type of CDG.
Represented on this slide are the first through 99th percentile of the normal reference ranges of each reported ratio, as established by our CLIR bioinformatics software.
I’d like to now go through some representative profiles that will highlight the utility of the testing offered here at Mayo Clinic.
ALG1-CDG, is a CDG in which a type I pattern is seen in the transferrin profile, indicating an assembly or transfer defect. The transferrin profile here shows an abundance of di-sialo transferrin, a higher than normal level of a-sialo transferrin, and lower levels of the fully glycosylated transferrin. The Apo-CIII profile is normal, and the total N-glycan analysis shows a signature glycan at the location m/z-1124.
SLC35A2-CDG, or early infantile epileptic encephalopathy-22, is a CDG in which a type II pattern is seen in the transferrin profile. The transferrin profile, as you can see, has several peaks representing partially glycosylated protein. This is a type II pattern and indicates a remodeling or processing defect. There is increased tri-sialo and mono-sialo transferrin contrasting with what is seen in a type I profile. Total glycan analysis reveals the characteristic signature glycans, and the Apo-CIII profile is non-informative.
Mixed CDGs have defects in either the assembly or transfer of the dolichol-linked glycan and in its processing. Seen here is a profile for PGM1-CDG. Again Apo-CIII is normal, but the transferrin has increased di-sialo transferrin along with increased number of partially glycosylated species. In addition, there are several abnormal glycans present in the N-glycan profile.
Here we have a CDG with an informative Apo-CIII profile but normal transferrin and N-glycan analysis(not shown here). Compared to the normal profile, a GALNT2 profile has a much higher a-sialo to di-sialo ratio. And the fully glycosylated Apo-CIII is low, indicating defective post-translational O-linked glycosylation of Apo-CIII.
And now I present a CDG with a biochemical profile showing abnormalities in the total N-glycan analysis only. Both Apo-CIII and the transferrin analysis are normal. The N-glycan analysis shows that the glycan at position 2605 is low in this patient relative to 2431, and the glycan at position 2966 is low relative to 2792. This highlights that glycans not seen, or seen in reduced quantity, can be diagnostic as well. This particular CDG, SLC35C1-CDG, is a member of a group of disorders with a defect in the processing of protein-bound glycans.
And finally, to complete the description of possible abnormalities, here we have an informative urine oligosaccharides analysis in the setting of normal blood screening for CDG, as represented by MOGS-CDG. The abnormality seen has mass/z ratio of 990, and its accumulation in the patient's urine indicates a glycosylation defect — specifically, defective biosynthesis of N-linked oligosaccharides due to glucosidase I deficiency.
This group of disorders is large and continues to grow with gene discovery. In any unexplained disorder, especially when neurologic symptoms are present, one should always think to test for CDGs. The screening strategy we propose for CDG casts a wide net overall and is designed to pick up the largest number of cases, sort them for follow-up testing, and diagnose as efficiently as possible. Though many providers have started with a molecular panel or whole exome sequencing (WES) and sent to us for confirmatory biochemical testing, we do suggest starting with Mayo test code CDG. This will return one of four interpretations. Depending on the result and your level of suspicion for CDG, you can pursue one of several different avenues, culminating in a virtual complete work-up and possible diagnosis for a CDG. As a note, there are many CDGs that will not be picked up on any currently offered CDG screening test. If you continue to be strongly suspicious of a CDG following a negative biochemical and molecular workup, research options are available at select institutions.
The Mayo Clinic Congenital Disorders of Glycosylation Clinic offers coordinated care for individuals with a CDG. They collaborate closely with other departments, including Neurology, Cardiology, Ophthalmology, laboratory experts, and others, to ensure care is personalized to the needs of the patient. The clinic sees more patients with CDG than any other practice in the U.S., and our specialists are world experts. They continue to research and translate discovery to care for the best possible treatment for this rare condition.
The website address is listed below.
Mayo Clinic is also part of the Frontiers in CDG Consortium. There are 13 centers of excellence whose mission it is to leverage cross-disciplinary, team-based clinical science to address unresolved questions; to increase clinical trial readiness; to advance and share knowledge, awareness and education on CDG; and, most importantly, to develop treatments and meet currently unmet patient needs.
So, in summary, Mayo Clinic laboratory diagnostics and patient care for CDG is robust and continually improves to provide comprehensive care for those seeking a diagnosis and those already diagnosed with this rare condition.
