Human Molecular Genetics, 2002, Vol. 11, No. 5 599-604
© 2002 Oxford University Press
A frequent mild mutation in ALG6 may exacerbate the clinical severity of patients with congenital disorder of glycosylation Ia (CDG-Ia) caused by phosphomannomutase deficiency
The Burnham Institute, Glycobiology Program, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA, 1Department of Clinical Genetics, University Hospital Rigshospitalet, Copenhagen, Denmark, 2Center for Human Genetics, University of Leuven, Leuven, Belgium and 3Center for Metabolic Medicine, Great Ormond Street Hospital for Sick Children, Great Ormond Street, London WC1N 3JH, UK
Received November 28, 2001; Revised and Accepted January 7, 2002.
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ABSTRACT |
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Single nucleotide polymorphisms occur throughout the human genome. A gene that causes one of the congenital disorders of glycosylation (CDG) has a mutation (911T
C) that changes a phenylalanine to serine at position 304 (F304S) of the 
1,3 glucosyl transferase. We show that this change reduces the ability of the gene product to rescue defective glycosylation of an alg6-deficient strain of Saccharomyces cerevisiae during rapid growth. This finding suggested that the mutation might affect glycosylation in humans. We therefore compared the frequency of this variant in 301 controls and in 101 CDG patients who carry known mutations in other genes involved in CDG, i.e. PMM2 (CDG-Ia; 91 patients) and MPI (CDG-Ib; 10 patients). The variant allele frequency is identical in both CDG patients (0.30) and controls (0.28). Importantly, the F304S genotype frequency in 55 CDG-Ia patients classified as mild/moderate (n = 28), or severe (n = 27) was significantly higher in severely affected patients (0.41) than in mild/moderately affected patients (0.21). Mortality (n = 9) was higher when F304S was present (n = 6). Severely affected patients with the PMM2 mutations F119L/R141H (n = 22) carry the F304S mutation more often (0.36) than mildly affected patients (0.18, n = 11) with this mutation. Clinical severity of mildly affected sibs with the same PMM2 mutations did not correlate with F304S genotype. Thus, the presence of the F304S allele may exacerbate the clinical outcome, especially in severely affected CDG patients. We speculate that this type of variant may be implicated in other multi-factorial disorders that involve N-glycosylation. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutations in genes involved in N-glycan biosynthesis cause congenital disorders of glycosylation (CDG), a group of autosomal recessive metabolic disorders (for reviews see 1–3). The ALG6 gene encodes an
1,3-glucosyl transferase responsible for adding the first glucose to the Man9GlcNAc2-oligosaccharide chain linked to dolichol pyrophosphate (4). This step is required in order to fully mature the oligosaccharide and make it an optimal substrate for the oligosaccharide transferase complex linking the sugar chain to nascent proteins in the endoplasmic reticulum (5). Seven mutations in the ALG6 gene are currently known to cause CDG-Ic (6–10). One sequence variation, 911TC, causing a phenylalanine to serine substitution at amino acid position 304 (F304S), has previously been described to have a negative effect on 1,3-glucosyl transferase function, especially when combined with a mutation in WBP1, a component of the oligosaccharide transfer complex (10). CDG-Ic patients have dysmorphic features, floppiness, feeding problems, hypotonia, retarded motor development and recurrent seizures (11). Recently, Vuillaumier-Barrot et al. (12) reported that the variant F304S was not sufficient to cause CDG-Ic. We also encountered this variant while screening a small group of unclassified glycosylation-deficient patients for mutations in different genes that are known to cause CDG, and wondered whether the presence of this variant might influence the clinical outcome in patients with already impaired glycosylation. Most ‘simple’ Mendelian metabolic disorders are caused by recessive defects affecting both alleles of a single gene. However, this view does not account for the spectrum of clinical phenotypes within a specific disease, or the environmental conditions that exert considerable influence (13,14). Complex pathologies with clear genetic components are attributed to multiple defects in mostly unknown genes. The challenge is to identify them in the genetic background. The term synergistic heterozygosity was recently used to describe patients with partial defects in two or more genes in the same pathway (15). Analysis of Bardet–Biedl syndrome now shows that three mutant alleles in two different genes may be required for expression of the disease, blurring the distinction between mono- and poly-genic disorders (16,17).
We confirmed that this frequent variant in ALG6 compromises glycosylation in Saccharyomyces cerevisiae, and then compared its frequency in the general population to a cohort of CDG-I patients with mutations in other genes in the glycosylation pathway. F304S was almost twice as common in severely affected CDG-Ia patients as it was in patients listed as moderate/mild.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Identification of a functional variant in the ALG6 gene
Analysis of the ALG6 cDNA from a series of 10 undefined CDG patients revealed the presence of the 911T
C variant (F304S) in the heterozygous state of six patients and in the homozygous state of one patient. The presence of the F304S variant was confirmed by sequencing genomic DNA. These patients did not have additional mutations in the ALG6 gene. Further analysis identified mutations in other genes in the N-linked glycosylation pathway: 13 patients with mutations in PMM2, identifying them as CDG-Ia, and three patients with mutations in the MPI gene, classifying them as CDG-Ib, respectively. F304 is located in a transmembrane domain of the protein in an area that is not conserved between yeast and humans. However, other mutations in this area reduce glycosylation and are responsible for CDG-Ic (7,8). Changing a phenylalanine to a serine changes the volume of the amino acid from 135 Å3 to 73 Å3 (18,19), which might affect the function of the protein as the change at position 333 from alanine (67 Å3) to valine (105 Å3) is known to do (8). Thus, we investigated the functional effect of the amino acid change using a S.cerevisiae strain deleted for ALG6. Using the glycoprotein carboxypeptidase Y (CPY) as an endogenous model protein, we investigated the effect of the ALG6 mutation on glycosylation (20–22).
Recombinant cDNAs, encoding the wild-type and F304S variant proteins, were cloned into a yeast expression vector and transformed into an alg6-deficient strain with deficient N-linked glycosylation (23). As previously reported (6–8,10), normal human ALG6 is able to fully restore CPY glycosylation in the alg6-deficient S.cerevisiae strain. This is seen in cells taken from logarithmic (generation time 1.8 ± 0.2 h), late logarithmic (generation time 7 ± 1 h) or early stationary growth phase (generation time 18 ± 2 h) (Fig. 1, hALG6). In contrast, fast growing cells expressing the F304S encoding allele underglycosylate CPY and produce glycoforms lacking 1 and 2 N-linked chains. However, the effect of this allele is less dramatic when cells are growing more slowly (Fig. 1, F304S). Cells expressing the vector alone without any ALG6 result in the majority of CPY lacking oligosaccharide chains (Fig. 1, vector). To see if the F304S change produced a thermolabile glucosyl transferase, we also expressed the constructs at 37°C. This is not the optimal growth temperature for yeast and other factors such as heat shock responses (24–26) might affect glycosylation at the elevated temperature. However, the F304S change clearly affected glycosylation, since the ratio between normal and underglycosylated CPY was considerably lower in cells carrying the F304S allele. Also, the effect is more evident at 37°C than at 30°C, suggesting that the enzyme is thermolabile or that increased temperature makes glycosylation more sensitive to the ALG6 mutation. Quantitative measurements from four independent experiments of fast growing cells show a significant impact of the F304S variant. A greater portion of CPY lacks 1 and 2 N-linked chains compared to fully glycosylated CPY in the F304S complemented strain than in the strain expressing normal wild-type ALG6.
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Frequency of F304S in CDG-I patients and controls
After finding the 911T
C (F304S) variant in a relatively small group of unclassified patients, we wanted to investigate how frequently this mutation occurred in other known CDG patients compared to a series of healthy control subjects. We determined the frequency of the F304S allele in 91 confirmed cases of CDG-Ia and 10 confirmed cases of CDG-Ib. As shown in Table 1, the frequency in CDG patients (0.30) was essentially the same as a group of caucasian European and American controls (0.28). Both groups displayed Hardy–Weinberg equilibrium for the ALG6 genotypes. The frequency of the F304S mutation is significantly lower in Asian and African American populations (P.Kwok, personal communication).
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CDG-Ia patients with various clinical presentations
The next question was whether the presence of the F304S variant affected the clinical severity of CDG. For this analysis, we identified 55 CDG-Ia patients who were classified as mild (10), moderate (18) or severe (27) according to their clinical status (27). This group included seven sibships with mild to moderate presentations. There was no apparent correlation between the severity and the F304S genotypes among sibs with identical PMM2 mutations in the mild/moderate range (Table 2). The majority of these patients have the common severe R141H mutation along with another less frequent and generally milder mutation. Although ALG6 status has no obvious impact on this small group of non-severe patients, the results of F304S on yeast CPY glycosylation under stressful conditions led us to hypothesize that the effects of the F304S mutation might be more evident in severely affected patients. We therefore compared the frequency of the 911T
C allele in 27 severely affected patients to a group of patients considered as being mild/moderate (Table 3). The frequency in the severely affected group (0.41) was considerably higher than in the mild/moderate group (0.21), and the difference was highly significant (P = 0.0028) based on 
2 analysis. Six of the nine patients who died had the F304S genotype, but these numbers are too low for statistical significance. The increased frequency of 911TC in the severe group suggested that it might exacerbate their presentation. Since this group includes many different genotypes, we also compared the frequency of the 911TC allele in a subset of patients who all carry the R141H/F119L mutations in PMM2 (Table 3). These patients cluster at the severe end of the clinical spectrum. Patients from the Leuven group, two from North America and 20 previously categorized Danish patients were subdivided into most severe (22 patients) and less severe (11 patients) groups based on their clinical presentations and outcome. Again, the F304S genotype was about twice as frequent in the most severe group (0.36) compared to the less severe patients (0.18); however, the difference was not statistically significant (P = 0.076).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutations in different genes involved in N-glycosylation cause various types of CDG-I by reducing the availability of the precursor oligosaccharide for transfer to protein. Essentially all reported CDG cases have two mutations in a single affected gene leading to the disorder (10,28,29).
Digenic inheritance has been documented in other disorders (30–33). In those cases, individuals have mutations in two different genes involved in the same pathway. Both genes function at a heterozygous level since a normal allele is also present, but pathology occurs because both genes are central to the pathway. Recently, some cases of Bardet–Biedl syndrome were found to involve three mutated alleles in two different genes, blurring the distinction between strict Mendelian and multigenic inheritance (16,17).
Since most of the consequences of CDG are thought to result from hypoglycosylation of proteins, i.e. the same endpoint for each disease, the broad spectrum of clinical presentations is surprising. At the severe end of the spectrum, the most common PMM2 missense mutation (R141H) is probably lethal when homozygous (34,35). Most CDG-Ia patients presenting with a severe/classical clinical picture have 0–10% of normal PMM activity in fibroblasts and leukocytes (36–39) and their asymptomatic parents have 
50% residual activity. Increased awareness of CDG has lead to the identification of a group of CDG-Ia patients with milder phenotypes and borderline developmental delays. Biochemical investigations showed a substantial amount of residual PMM activity (27,40,41). Some of these patients have PMM activities in fibroblasts only slightly below the heterozygous level, but still show lower activities in leukocytes. Thus, other genetic factors might contribute to the clinical diversity of this disease.
In yeast, the consequences of ALG6 mutations depend on the presence of other mutations, e.g. WBP1 in the glycosylation pathway. In addition, environmental factors (different growth rates) substantially affect CPY glycosylation in F304S cells. We have shown here that the effects of this mutation are only evident when the glycosylation system must perform at highest efficiency. While it is difficult to directly extrapolate both genetic and environmental results from yeast to humans, a clear environmental effect was seen in one CDG-Ic patient with viral-induced gastroenteritis (7). This condition leads to protein-losing enteropathy (PLE), and loss of heparan sulfate (HS) from the basolateral surface of rapidly dividing small intestine enterocytes. When PLE resolves, the rate of cellular proliferation decreases and much of the HS reappears. It is significant that the more slowly dividing epithelial cells lining the esophagus, stomach and colon do not lose HS, even during periods of PLE. The already compromised glycosylation system appears overwhelmed during gastroenteritis because the localized infection increases enterocyte proliferation in this young child (7).
The allelic frequency of F304S among the controls and CDG-Ia patients was quite high and essentially identical, being 0.28 and 0.30, respectively (Table 1). Hardy–Weinberg equilibrium is respected in the patient group, and this underscores the fact that the deleterious mutations on both alleles of the disease gene (PMM2) are the primary determinants for the clinical presentation. Thus, we have excluded the possibility that these patients with mutations in PMM2 would not show up unless their glycosylation is further compromised by other defects in this pathway. In addition though, the significantly greater frequency of F304S among the severely affected patients points to ALG6 as a genetic modifier, and this allele in particular. Its impact appears to be greatest in patients who carry the most serious mutations in PMM2. This may simply reflect the contribution of further stress on an already debilitated N-glycosylation system.
In summary, we have shown that a common and relatively mild mutation in ALG6 can be found equally in CDG-I patients and controls, and it may exacerbate an already severe CDG phenotype. In addition, ALG6 mutations could be important in other multi-factorial diseases (42,43). We hypothesize that such variants in glycosylation, like the one we explored in the study, possibly contribute to the genetic background in other more common diseases where glycosylation could play a role in the severity of the disease.
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MATERIALS AND METHODS |
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Media and materials
Most of the materials were obtained from Sigma Chemical Co. (St Louis, MO) except for the following: minimal essential medium (
-MEM and DMEM) (Gibco BRL, Baltimore, MD), RPMI 1604 medium (Irvine Scientific, La Jolla, CA), fetal bovine serum (Hyclone Laboratories, Logan, UT). Restriction enzymes and T4 DNA ligase were from Promega (Madison, WI), AmpliTaq DNA polymerase was from Roche Diagnostics (Berkeley, CA). 32P-
ATP, used to 5'-label the oligonucleotides for dot blot analysis, was from Amersham Pharmacia Biotech (Roosendaal, the Netherlands). Oligonucleotides were from Genbase (San Diego, CA) and Eurogentec (Herstal, Belgium). Sequencing was performed using BigDye sequencing kit on an ABI 377 DNA sequencer, both from Applied Biosystems (Foster City, CA). S.cerevisiae and Escherichia coli were grown in standard YPD, SC and LB media (44,45).
Analysis of the cDNA and genomic DNA
Total RNA was extracted using the RNAeasy kit (Qiagen). First-strand cDNA of the human ALG6 was synthesized essentially as described (7). The mutations found in the cDNA were confirmed by sequencing genomic DNA. Purification of genomic DNA from patients was done essentially as described previously (46). Control DNA was from Coriell’s DNA polymorphism discovery resource (415 subjects) and from an anonymous Belgian control population (221 subjects). The 911TC (F304S) mutation is located in exon 10 of the ALG6 gene (10). To amplify and sequence ALG6 exon 10, oligonucleotides oVW218, 5'-AAA CTT AAG TTG ATA AAT AAT ATG ATC CTT-3'; and oVW219, 5'-GTC TAA CAC AGA AGC TAA GTA TGG G-3' were used in the following PCR cycle: 95°C for 3 min, 35x (94°C for 20 s, 56°C for 30 s, 70°C for 45 s), then 70°C for 7 min. Purification and sequencing of the products were either performed as described by Kim et al. (47) or analyzed essentially as described by Chen et al. (48).
The frequency of the 911TC mutation in the Belgian control populations and in the ‘Leuven’ cohort of CDG patients (27,29) was determined using a dot blot assay. In brief, exon 10 of the ALG6 gene was amplified using primers 5'-AAACTTAAGTTGATAAATAATATGATCCTT-3' and 5'-AAAAGATCAGTTGTGGCAAGA-3' in a ‘touch-down’ PCR cycle: 94°C for 5 min; 20x –0.5°C/cycle (94°C for 20 s, 65°C for 45 s, 72°C for 30 s); 26x (94°C for 20 s, 55°C for 30 s, 72°C for 30 s); 72°C for 7 min. Five microlitres of the PCR products were blotted onto a N+-hybond membrane (Amersham-Pharmacia-Biotech). Wild-type specific (5'-AACGTAAAACAAAAGCTC-3') and mutant specific (5'-GAGCTTTTGTTCTACGTT-3') probes were 5'-labeled using [-32P]ATP and T4 PNK as described (45). Hybridizations with the labeled probes were performed in 6x SSC (sodium chloride/sodium citrate buffer), 5x Denhardt’s, 0.5% SDS and 200 µg/ml heparin at 42°C (45). Blots were washed in 2x SSC and 0.5% SDS at 45°C (wild-type oligonucleotide) and 50°C (mutant oligonucleotide).
Strains and plasmids
The ALG6-deficient S.cerevisiae strain YG227 (Mat ade2-101 ura3-52 his3
200 lys2-801 alg6::HIS3) (23) was used to express the human 1,3 glucosyltransferase gene ALG6. Subcloning and transformation of E.coli and yeast were carried out using standard procedures (45). The plasmids expressing normal hALG6 and F304S hALG6 were constructed by generating specific restriction enzyme sites at the 5' and 3' end of the cDNA as previously described (6,7) and using the expression vector pWE85 (7) as vector. All plasmids were re-sequenced before transformation into S.cerevisiae strain YG227.
Analyzing the effect of the ALG6 mutation
Saccharomyces cerevisiae strain YG227 (23) containing one of the following plasmids: pWE85 (without the ALG6 gene), pWE187 (containing wild-type human ALG6 cDNA), or pWE183 (encoding F304S 1,3 glucosyl transferase) was used to investigate the effects of the mutation in ALG6 on the in vivo glycosylation of CPY. The transformed yeast cells were grown in synthetic complete medium without uracil (SC-URA) (44) overnight at 30°C. The next day the cells were diluted to an optical density at 600 nm of 0.05 and followed spectrophotometrically. Cells were harvested at various growth phases, OD600 < 0.3, OD600 = 1.0 and OD600 
2.0 by centrifugation, and analyzed as described previously (7). The intensity of the various CPY glycoforms seen on the western blot was quantified using AlphaEase 4.0 from Alpha Innotech Corporation (San Leandro, CA).
Clinical description and assessment of CDG patients
Genotype–phenotype correlation in PMM2-deficient CDG-Ia patients has not been demonstrated, although several studies have described mutations and categorized patients based on their clinical severity. We selected a group of previously described European CDG-Ia patients (Leuven Cohort) (50) with a variety of PMM2 mutations who were categorized as having mild, moderate or severe clinical presentations. In addition, 11 North American CDG-Ia patients with various PMM2 mutations were categorized by the same criteria. In total, 10 mild and 18 moderately affected patients were combined into one group and compared to 27 severely affected patients. In addition, a series of 20 severely affected previously described Danish patients (49), two American patients, and patients from the Leuven cohort who carried the F119L/R141H mutations in PMM2, were subdivided into less and most severe based on their functional outcome.
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ACKNOWLEDGEMENTS |
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Drs Ming Xiao and Pui-Yan Kwok are gratefully acknowledged for analyzing American caucasian control DNA samples. Prof. Markus Aebi is gratefully acknowledged for the
alg6 yeast strain and Dr Jakob Winther for the CPY antibody. We appreciate the expertise and help from D.Foster from the ‘Gene Analysis’ facility at The Burnham Institute and thank Patty Lucker for excellent technical assistance. This work was supported by grants from the ‘CDG Parents Network Foundation’, March of Dimes Grant FY99-205 and NIH ROI DK55615 to H.H.F., biotechnology research and training grant from the University of California, San Diego to V.W., and grant G.0243.98 from the Flanders Fund for Scientific Research to G.M.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 858 646 3142; Fax: +1 858 713 6281; Email: hudson@burnham.org Present address: Vibeke Westphal, Novo Nordisk, Novo Allé 6As.068, DK-2880 Bagsvaerd, Denmark
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