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Congenital Disorder of Glycosylation Id Presenting with Hyperinsulinemic Hypoglycemia and Islet Cell Hyperplasia

The Burnham Institute (L.S., E.A.E., C.W., H.H.F.), Program for Glycobiology and Carbohydrate Chemistry, La Jolla, California 92037; and Columbia University Departments of Pediatrics (W.K.C.) and Pathology (J.C.), New York, New York 10032

Address all correspondence and requests for reprints to: Hudson H. Freeze, The Burnham Institute, Program for Glycobiology and Carbohydrate Chemistry, 10901 North Torrey Pines Road, La Jolla, California 92037. E-mail: hudson{at}burnham.org.

	Abstract

Top Abstract Introduction Patient and Methods Results Discussion References

 
Context: Inborn errors in protein glycosylation, such as the congenital disorders of glycosylation (CDGs), generate multifaceted syndromes that impair many organ systems. We here report the diagnosis of the third known patient with CDG-Id.

Results: The patient’s phenotype was extremely severe, and she succumbed at 19 d of age. Leading features included hyperinsulinemic hypoglycemia, and autopsy revealed islet cell hyperplasia with increased ß-cell mass. Other features were a Dandy-Walker malformation, facial dysmorphisms, and profound hypotonia. The patient carried a novel homozygous point mutation (512G>A) in the hALG3 gene, which encodes a mannosyltransferase. Lentiviral complementation with wild-type hALG3 corrects the biochemical defect in the patient’s fibroblasts.

Conclusions: Our findings underscore the importance of proper glycosylation in several major organ systems and emphasize that CDG should be ruled out in patients with persistent hyperinsulinemic hypoglycemia of unknown etiology.

	Introduction

Top Abstract Introduction Patient and Methods Results Discussion References

 
CONGENITAL DISORDERS OF glycosylation (CDGs) are inherited metabolic syndromes with defective biosynthesis or transfer of lipid-linked oligosaccharides (LLOs) to the nascent protein chain (type I) or compromised processing of protein-bound oligosaccharides (type II; for review see Ref. 1). Most known cases belong to type I, which, to date, has 12 defined subgroups (CDG-Ia to CDG-IL). Except for CDG-Ia-c, there are very few (less than 10) identified cases in each subgroup. Common symptoms of the CDG type I group include developmental delay, hypotonia, cerebellar hypoplasia, esotropia, failure to thrive, and coagulopathy. An exception is CDG-Ib, which shows no neuronal involvement (1, 2, 3). Hyperinsulinemic hypoglycemia (HH) has been described in several cases, mostly in CDG-Ib (4, 5), but also as the leading symptom in a CDG-Ia patient (6). The molecular explanations for this dysregulation of insulin release are unknown, but hypoglycosylation of the sulfonylurea receptor-1 (SUR1) has been speculated to be involved. The previously described patients have, however, been responsive to diazoxide treatment, indicating the presence of at least some functional ATP-sensitive K⁺ channels on the cell surface (6).

CDG-Id (OMIM 601110; formerly known as CDGS type IV) is caused by mutations in the human orthologue of yeast alg3, hALG3 (7). This gene encodes dolichyl-P (Dol-P)-mannose (Man):Man₅-N-acetyl glucosamine (GlcNAc)₂-PP-Dol--1,3-mannosyltransferase, the enzyme catalyzing the addition of the sixth Man residue to the growing LLO chain (7). At present, only two CDG-Id patients have been reported (7, 8), and a clinical description is available for only one of them. This patient showed severely delayed psychomotor development; abnormal muscle tone (initially spastic, later dystonic tetraparesis); atrophies of the cerebellum, cerebrum, corpus callosum, and optic nerve; intractable seizures; coloboma of the iris; and dysmorphisms. The liver function was normal, and no signs of coagulopathy were noted (7, 9). Nothing was noted about glucose (Glc) dysregulation in this patient.

The molecular etiology for these two cases has been delineated. The first patient was homozygous for a 353G>A mutation, yielding the amino acid substitution G118D (7). The second patient carried a homozygous 165C>T mutation in exon 1, activating a cryptic splice site that results in the deletion of 37-bp of coding sequence and the generation of a premature stop codon (8).

Here we outline the clinical and molecular findings of the third known CDG-Id patient, including a comprehensive clinical description, molecular diagnosis, and phenotypic rescue using lentiviral transduction. We also provide evidence for the cause of the HH in this patient.

	Patient and Methods

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Materials

-MEM, DMEM, antibiotics (penicillin/streptomycin), L-glutamine, and TRIzol reagent were all from Invitrogen (Carlsbad, CA). Fetal bovine serum and bovine growth serum were from HyClone (Logan, UT), [2-³H]Man (20 Ci/mmol) from PerkinElmer (Boston, MA), the Microsorb-MV NH₂ HPLC column from Varian Medical Systems, Inc. (Walnut Creek, CA), and oligonucleotides from GenBase Solutions Ltd. (San Diego, CA). All other chemicals were purchased from Sigma Aldrich (St. Louis, MO) and were of analytical grade.

Approval of human research

Approval of human research was obtained from the institutional review boards of Columbia University (New York, NY) and The Burnham Institute (La Jolla, CA).

Immunohistochemical staining and morphometric analysis

Immunohistochemical staining was performed according to standard methods after deparaffinization using an automated immunohistochemical staining machine (Autostainer Plus; DAKO Corp., Carpinteria, CA). Slides were immersed in citric buffer at pH 6.0 and boiled for 7 min at full power. They were then boiled for 10 min at 40% pressure. Staining for insulin was performed using a primary mouse monoclonal antibody (BioGenex Laboratories, Inc., San Ramon, CA) at a 1:50 dilution and incubated for 40 min. Secondary labeling and visualization were performed using DAKO Corp. Envision⁺ System for an incubation period of 30 min. Morphometric analysis was performed using Image Pro Plus Image Analysis Software (Media Cybernetics, Silver Spring, MD), and statistical significance was determined using a one-tailed t test. Pancreatic sections from the subject were compared with two age-matched controls, demonstrating no evidence of abnormal glucose homeostasis.

Cell culture

After obtaining written informed consent, a fibroblast culture was established from a skin biopsy from the patient. The cells were maintained as described (10).

LLO analysis

[2-³H]Man-labeled LLOs from subconfluent fibroblasts were prepared as described (10). The oligosaccharides were released using mild acid hydrolysis (0.1 M HCl) and separated by amine adsorption analysis HPLC on a Microsorb-MV NH₂ column using a gradient of 65–35% acetonitrile. 2-Aminobenzamide-labeled Man_5–9GlcNAc₂ oligosaccharides (Prozyme, San Leandro, CA) were used as internal standards.

Lentiviral complementation

Wild-type cDNA copies of hALG3, DPM1 (encoding Dol-P-Man synthase; deficient in CDG-Ie), and MPDU1 (encoding Dol-P-Man/Dol-P-Glc utilization defect 1; deficient in CDG-If) were cloned into lentiviral vector pLenti6/V5-D-TOPO (Invitrogen), and became LV-hALG3, LV-DPM1, and LV-MPDU1, respectively. LV-GFP is a control lentiviral vector that contains the GFP gene. Lentiviruses were produced in 293FT cells as follows: 6 x 10⁶ 293FT cells in a 10-cm tissue culture plate were transfected with 9 µg of the ViraPower Packaging Mix (Invitrogen) and 3 µg lentiviral plasmid via the Lipofectamine transfection method. Next day, the medium was replaced with fresh DMEM containing 10% FBS. Lentiviruses were harvested 48–72 h post transfection. Fibroblasts were transduced by lentivirus and labeled for LLO analysis 72 h after infection. A lentivirus containing only the GFP gene was used as control.

Analysis of hALG3 cDNA and genomic DNA

Total RNA was isolated from cultured fibroblasts with TRIzol Reagent (Invitrogen). hALG3 cDNA was amplified with SuperScript One Step RT-PCR kit (Invitrogen). The primers used for RT-PCR were: 5'-GGTGGGCCCACACAAGCGGCG-3'; and 5'-GACTCAGGTCCTGAGGGA AA-3'. RT-PCR cycling condition was 50 C, 40 min; 94 C, 2 min; 40x (94 C, 30 sec; 55 C, 30 sec; 72 C, 90 sec). Genomic DNA was isolated from fibroblast with the Wizard Genomic DNA Purification Kit (Promega Corp., Madison, WI). The primers used to amplify hALG3 exon 4 were 5'-GCAAGGTGAGTCCATGCTAC-3'; and 5'-TTTCTATAGATCCTGGACTC-3'. The PCR conditions were the following: 94 C, 3 min; 30x (94 C, 30 sec; 50 C, 30 sec; 70 C, 45 sec); 70 C, 10 min.

	Results

Top Abstract Introduction Patient and Methods Results Discussion References

 
Patient description

Our patient, KR, was born after 36 wk gestation via normal spontaneous vaginal delivery to a 29-yr-old G2P1 mother. At 29–31 wk gestation, the fetus was diagnosed by ultrasound and subsequent MRI to have shortened femurs measuring at the fifth centile, complete agenesis of the corpus callosum, Dandy Walker malformation with a dilated fourth ventricle, absent cerebellar vermis with small cerebellar hemispheres, enlarged cisterna magna, a posterior fossa cyst, bilateral hydronephrosis and hydroureters, bilaterally flexed wrists and fingers, and oligohydramnios. Maternal serum screening was normal, and prenatal karyotype was 46, XX. There was no known history of consanguinity, but both parents were from the Dominican Republic.

The birth weight was 3.25 kg (25–50%), with a length of 50.0 cm (50%), and head circumference of 31 cm (<3%). The Apgar score was 2 at 1 min, and 8 at 5 min. Physical examination at birth demonstrated dysmorphic features, including thickened, large, low-set ears with abnormally shaped pinnae; unilateral posterior ear creases; flattened nasal bridge; thin upper lip; and micrognathia (Fig. 1, A and C). All four limbs were short, and the fingers and toes were long and thin with flexion contractures of the wrists, knees, and fingers. Toes were overlapping on one foot. The nipples were widely spaced and inverted, and the buttocks and the anterior and posterior neck showed abnormal fat distribution. There was a large irregularly shaped hemangioma over the buttocks and the posterior right leg. The baby had a weak cry and poor suck and was lethargic, with few spontaneous movements and severe hypotonia. Ophthalmologic examination demonstrated bilateral optic nerve atrophy with poorly reactive pupils.

View larger version (102K): [in this window] [in a new window]   FIG. 1. Postmortem presentation of the patient. A, Close-up of the patient’s head and neck shows microcephaly; thickened, large, low-set ears with abnormally shaped pinnae; unilateral posterior ear creases; flattened nasal bridge; thin upper lip; micrognathia; and abnormal fat distribution around the neck. B, A radiograph demonstrates short vertebral bodies, ribs, radii and ulnae, femurs, tibiae, and fibulae; paddle-shaped iliac wings; and bilateral small scapulae. C, Whole-body examination shows, apart from facial and neck dysmorphisms, inverted wide-spaced nipples; shortened limbs; flexed wrists, fingers, and knees; and long fingers and toes.

The patient’s clinical course was complicated by recurrent hypoglycemia (less than 2.2 mmol/liter) requiring high concentrations of dextrose infused at 15 mg/kg/h. Hyperinsulinemia (19.9 µU/ml) was present during the hypoglycemic episodes, and ß-hydroxybutyrate was suppressed (0.02 mmol/liter) (reference interval, 0.06–0.17). Thrombocytopenia was persistent, with platelet counts of 23,000–71,000. Creatine kinase was persistently elevated (662–1,483 U/liter); transaminases were mildly elevated, with AST of 55–283 U/liter and ALT of 25–66 U/liter; and liver synthetic function was diminished, with total protein of 3.5 g/dl, albumin of 1.7 g/dl, and prolonged coagulation with a prothrombin time of 22.1 sec and a partial thromboplastin time of 79 sec. She required gavage feeding due to poor suck and swallow. Ultimately, she became hemodynamically unstable on day of life 19 and died of urosepsis with Enterobacter cloacae.

Postmortem examination demonstrated multiple dysmorphisms on gross examination (Fig. 1, A and C). The lungs exhibited incomplete lobation bilaterally. The cardiac ventricles were bilaterally hypertrophied, with the thickness of the right and left ventricles measuring 0.4 and 0.7 cm, respectively. The liver was large and had several well-defined lobular structures protruding from its anterior surface. The cut hepatic surface was pale and contained dilated bile lakes. The spleen contained a fissure along its entire length. The reproductive system contained a double-barrel vagina and a bi-cornuate uterus. The brain demonstrated a Dandy-Walker malformation, agenesis of the cerebellar vermis, and agenesis of the corpus callosum.

Histologically, the pancreas exhibited marked increased islet numbers, demonstrated by widespread immunohistochemical staining for insulin (Fig. 2; see below). The heart contained dilated sinusoids lined by endothelial cells, and sections from the lungs contained dilated lymphatic spaces. The liver showed bile duct plate malformations, defined by the persistence of an excess of embryological bile duct structures in ductal plate configuration. Moderate to severe steatosis and grade 1–2/4 periportal hepatocellular hemosiderosis were also demonstrated.

View larger version (161K): [in this window] [in a new window]   FIG. 2. Insulin immunohistochemistry of the patient’s pancreas indicates islet cell hyperplasia. Sections from the patient’s pancreas (left) and an age-matched control (right) are stained for insulin using immunohistochemistry. The patient shows pancreatic islet ß-cell hyperplasia, together with enhanced insulin staining.

Formalin-fixed, paraffin-embedded pancreatic tissue from this case, as well as two age-matched controls, were immunohistochemically stained for insulin. Insulin staining for ß-cells as a proportion of the total pancreatic area demonstrated was 3-fold higher in our case (4.2% in the case vs. 1.1% in two controls). The average ß-cell size was 21% larger in our case, compared with the controls, indicating modest ß-cell hypertrophy. The average islet size was comparable between our case and the controls. However, the majority of the increase in total ß-cell mass was due to an increase in the total number of islets, which was 5.8 times greater in our case, relative to the two controls.

Analysis of glycosylation

The symptoms were suggestive of CDG, and carbohydrate-deficient transferrin analysis by electrospray ionization-mass spectrometry was performed (Mayo Clinic, Rochester, MN). This quantifies the relative amounts of the differently glycosylated transferrin isoforms, carrying zero, one, or two oligosaccharides. The ratio of transferrin molecules carrying one sugar chain/two sugar chains was 0.437 (normal 0.074), whereas the ratio of nonglycosylated transferrin to those with two sugar chains was normal. The activities of phosphomannomutase (deficient in CDG-Ia) and phosphomannose isomerase (deficient in CDG-Ib) were normal (data not shown). Next, the synthesis of LLOs in patient fibroblasts was assessed. The fibroblasts were found to accumulate the truncated LLO species Man₅GlcNAc₂-P-P-Dol (Fig. 3A, upper chromatogram), whereas a control cell line produced mainly the full-size Glc₃Man₉GlcNAc₂-P-P-Dol (Fig. 3A, middle chromatogram).

View larger version (18K): [in this window] [in a new window]   FIG. 3. Assessment of patient’s LLO production and mutational analysis of hALG3. A, [2-3H]Man labeling of the patient’s fibroblasts reveals accumulation of the truncated oligosaccharide Man5GlcNAc2 (upper chromatogram), whereas a control fibroblast cell line mainly produces the full-size version Glc3Man5GlcNAc2 (middle chromatogram). Patient fibroblasts produce full-size LLO upon transduction with a lentivirus carrying the wild-type hALG3 gene (lower chromatogram). Standard elution points for Man5GlcNAc2 (M5), Man9GlcNAc2 (M9), and Glc3Man9GlcNAc2 (G3M9) are indicated with arrows. B, Analysis of the genomic DNA from the patient shows the presence of a homozygous point mutation (512G>A) in exon 4 of hALG3 (upper chromatogram). Genomic DNA from the patient’s mother shows a heterozygous mutation (middle chromatogram), whereas DNA from a control is normal (homozygous for 512G) in this position (lower chromatogram).

At least six genes are involved in this biosynthetic step of the LLO synthesis (Man₅GlcNAc₂ Man₆GlcNAc₂), and three have been shown to cause different types of CDG. We transduced patient cells with a panel of four lentiviral constructs, either GFP alone (control) or GFP together with one of these potentially defective genes (hALG3, DPM1, or MPDU1, respectively). Seventy-two hours after transduction, the LLO production of the cells was investigated. Only cells transduced with hALG3 produced normal-sized LLOs (Glc₃Man₉GlcNAc₂-P-P-Dol; Fig. 3A, lower chromatogram), whereas cells transduced with the control lentivirus or lentiviral constructs expressing DPM1 or MPDU1 (data not shown) produced truncated versions.

Analysis of hALG3 in the patient and her mother

Mutational analysis of the patient hALG3 cDNA revealed a homozygous point mutation (data not shown) that causes the amino acid substitution R171Q. The mutation was confirmed on genomic DNA level (512G>A; Fig. 3B, upper chromatogram). The mother of the patient was found to be heterozygous for the mutation (Fig. 3B, middle chromatogram), but DNA was not obtainable from the patient’s father. No other mutations were found. However, as has been previously published (8), a substantial portion of the mRNA transcripts contained a 37-bp deletion (del160–196) both in patient and control.

	Discussion

Top Abstract Introduction Patient and Methods Results Discussion References

 
CDG is a rapidly growing group of disorders with a wide spectrum of symptoms and severity. Most subtypes have been discovered within the last 5 yr, and the number of known patients within each subtype is, except for CDG-Ia-Ic, equal to or less than five. This probably grossly underestimates the true incidence, because CDG is not considered a differential diagnosis unless classical signs are present, such as inverted nipples, abnormal fat distribution, and cerebellar hypoplasia. Our patient exhibited a wide variety of symptoms from several organ systems (see Table 1 for comparison with other CDG-Id patients and classical CDG-Ia and CDG-Ib), including HH and islet cell hyperplasia. Persistent HH as the leading symptom has been reported in both CDG-Ia and -Ib (6, 11), but not in any other CDG subtype, and we thus expand the finding of HH to CDG-Id. The reason for the insulin dysregulation in these cases is unknown. A feasible explanation is based on findings by Conti et al. (12), who showed that the SUR1 needs proper glycosylation to exit the Golgi apparatus. So-called trafficking mutations in SUR1 are known to cause HH (13, 14), and the finding of islet hyperplasia in the postmortem exam could also be explained by a lack of functional SUR1 (15). Most CDG HH patients, however, have been sensitive to diazoxide treatment, which strongly indicates the presence of some functional SUR1/Kir6.2 hetero-octamers on the surface. This is consistent with the finding that most CDG-I patients still glycosylate a substantial portion of their glycoproteins normally. Most CDG patients with HH are type Ib (phosphomannose isomerase deficiency), which is treatable by alimentary supplementation with Man (16). Taken together with the fact that HH was found in a third CDG subtype, we suggest that CDG always should be excluded, either by electrospray ionization-mass spectrometry or isoelectric focusing of transferrin, in patients with HH of unknown etiology. This extends the previous recommendation by Dekelbab and Sperling (14) that CDG-Ib should be excluded when HH is present together with protein-losing enteropathy and liver dysfunction.

	Acknowledgments

 
Mrs. Mie Ichikawa is greatly appreciated for initial experiments including analysis of the phosphomannose isomerase and phosphomannomutase activities. We are grateful to Rokuro Ito for pancreatic image analysis.

	Footnotes

 
This work was supported by National Institutes of Health Grant R01DK65615 (to H.H.F.), a postdoctoral fellowship from STINT/VR (Sweden; K2004–99PK-14887–02B) (to E.A.E.), and grants from the Children’s Cardiomyopathy Foundation and the Russell Berrie Foundation (to W.K.C.).

First Published Online April 19, 2005

¹ L.S. and E.A.E. contributed equally to this work and should both be considered first authors.

Abbreviations: CDG, Congenital disorder of glycosylation; Dol, dolichyl; Glc, glucose; GlcNAc; N-acetyl glucosamine; HH, hyperinsulinemic hypoglycemia; LLO, lipid-linked oligosaccharide; Man, mannose; SUR, sulfonylurea receptor.

Received February 3, 2005.

Accepted April 12, 2005.

	References

Top Abstract Introduction Patient and Methods Results Discussion References

 

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sun, L. Articles by Freeze, H. H. Search for Related Content PubMed PubMed Citation Articles by Sun, L. Articles by Freeze, H. H. Related Collections Pediatric Endocrinology Metabolism