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ALG1-CDG: A new case with early fatal outcome
Gene 534 (2014) 345–351
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ALG1-CDG: A new case with early fatal outcome A.-K. Rohlfing a,1, S. Rust b, J. Reunert a,1, M. Tirre b, I. Du Chesne a,1, Sa. Wemhoff c, F. Meinhardt c, H. Hartmann d, A.M. Das d, T. Marquardt a,⁎,1 a
Universitätsklinikum Münster, Klinik und Poliklinik für Kinder- und Jugendmedizin-Allgemeine Pädiatrie, Münster, Germany Leibniz-Institut für Arterioskleroseforschung, Münster, Germany Institut für Molekulare Mikrobiologie und Biotechnologie, Münster, Germany d Clinic for Pediatric Kidney, Liver Metabolic Diseases, Hannover Medical School, Hannover, Germany b c
a r t i c l e
i n f o
Article history: Accepted 8 October 2013 Available online 21 October 2013 Keywords: Congenital disorder of glycosylation type Ik Hypoglycosylation ALG1 pseudogenes Seizures Hypoproteinemia
a b s t r a c t 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 N C (M382T) and c.1312C N 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. © 2013 Elsevier B.V. All rights reserved.
1. 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 Abbreviations: ALG1, Asparagine-linked glycosylation 1 homolog (yeast, beta-1,4mannosyltransferase) = chitobiosyldiphosphodolichol beta-mannosyltransferase; CDG, Congenital disorders of glycosylation; GDP, Guanosine diphosphate; Man, Mannose; RER, Rough endoplasmic reticulum; HMT1, Human mannosyltransferase 1; GlcNAc, N-acetyl-D-glucosamine; PP, Pyrophosphate; kDa, Kilodaltons; CDT, Carbohydrate deficient transferrin; IEF, Isoelectric focusing; IMPP, Immunoprecipitation; SDS, Sodium dodecyl sulfate; PAGE, Polyacrylamide-gel electrophoresis; HPLC, High performance liquid chromatography; MEM, Minimum essential medium; PBS, Phosphate buffered saline; LLO, Lipid-linked oligosaccharides; DMEM, Dulbecco's Modified Eagle's Medium; cDNA, DNA complementary to RNA; EDTA, Ethylenediaminetetraacetic acid; PCR, Polymerase chain reaction; dNTP, Deoxyribonucleoside triphosphate; DMSO, Demethylsulfoxide; CPAP, Continuous positive airway pressure; CK, Creatine kinase; CK-MB, Creatine kinase muscle-brain, i.e. myocardial subtype of CK; CRP, C-reactive protein (reacting with C-polysaccharide of Pneumococcus); IgG, Immunoglobulin G; Glc, Glucose; CEPH, Centre d'Etude du Polymorphisme Humain; DPAGT1, Dolichyl-phosphate (UDP-N-acetylglucosamine) N-acetylglucosaminephosphotransferase 1; IGF1, Insulin-like growth factor 1; IGFBP3, Insulin-like growth factor-binding protein 3; ALT, Alanine-aminotransferase; GPT, Glutamic-pyruvic transaminase; AST, Aspartate aminotransferase; GOT, Glutamic oxaloacetic transaminase; GLDH, Glutamate dehydrogenase; CHE, Cholinesterase; AT-III, Antithrombin-III. ⁎ Corresponding author at: Department of Pediatrics University Hospital of Münster Albert-Schweitzer-Campus 1 48149 Münster, Germany. E-mail address: [email protected] (T. Marquardt). 1 Tel.: +49 251 8358519; fax: +49 251 8356085. 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.10.013
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-PPdolichol mannosyltransferase (alias chitobiosyldiphosphodolichol betamannosyltransferase; 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
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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 ALG1CDG. 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. 2. Materials and methods 2.1. 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). 2.2. 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 10% fetal bovine serum (Invitrogen/Life Technologies, Darmstadt, Germany), 2mM L-glutamine, 100 μg/ml penicillin and streptomycin. For subsequent RNA- and DNAextractions fibroblasts were obtained by trypsin treatment and washed in phosphate buffered saline (PBS) (PAA) before they were resuspended in 200 μl 0.9% NaCl and stored at −80 °C (Würde et al., 2012). 2.3. LLO analysis Fibroblasts from the patient and control cells were labeled with 100 μCi [2-3H]-mannose per ml labeling medium (DMEM without glucose: MEM, 9:1) for 45 min, incubated in MEM for 10 min, and subsequently washed with PBS as described previously (Chantret et al., 2003). The metabolically labeled dolicholpyrophosphate-linked oligosaccharides were extracted, purified and analyzed using high performance liquid chromatography (HPLC) as described (Kranz et al., 2001). 2.4. Mutation analysis RNA was isolated from control and patient fibroblasts by using the Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany). For generating cDNA, RNA was transcribed with SuperScript™ III Reverse Transcriptase (Invitrogen/Life technologies) and oligo (dT) primers (Fermentas, St. Leon-Rot, Germany). EDTA blood samples were taken from the patient and his parents to isolate genomic DNA with QIAmp DNA mini kit (Qiagen). The ALG1 coding sequence (NM_019109.4) was amplified with specific primers and PCR conditions (Supplementary Table 1). The amplified PCR products were purified with the PCR Product PreSequencing Kit (USB Products/Affymetrix, Ohio, USA). Sequencing was performed using BigDye Terminator Kit 3.1 (Applied Biosystems/Life technologies, Darmstadt, Germany) and the primers used for PCR under the following conditions: 96 °C for 2 min followed by 25 cycles at 94°C for 10s, at 50°C for 5s and at 60°C for 2min. Samples were purified using Sephadex/Millipore System (GE Healthcare, Buckinghamshire, UK/Merck Millipore, Schwalbach, Germany), and analyzed on an ABI PRISM 3730 sequencer. For confirmation of the patient’s ALG1 mutations at the genomic level, exon 11 and 13 were amplified (primers and PCR conditions are listed in Supplementary Table 1). Specific primers were designed to differentiate between the ALG1 gene (NG_009202.1) and its documented pseudogenes. PCR products were purified and sequenced as described above using universal primers (Supplementary Table 1).
2.5. Investigation of the mutation c.1312C N T in exon 13 In order to make sure that the heterozygous mutation c.1312C N T in the patient’s DNA was not only an artifact due to amplification of pseudogene sequences homologous to ALG1, as suggested in (Morava et al., 2012), the genomic ALG1 sequences of the patient and a healthy control were amplified under more stringent conditions using the HotStarTaq DNA Polymerase (Qiagen) and primers ALG1-ex13F and ALG1-ex13R. The Master Mix also contained 10× PCR Buffer (Qiagen), 5× Q-Solution (Qiagen), MgCl2 (Qiagen), A. dest. and dNTPs (GE Healthcare). For PCR amplification, samples were incubated at 94 °C for 15 min followed by 35 cycles of 94 °C for 30 s, 65 °C for 30 s, and 72 °C for 30 s, and a final incubation at 72 °C for 7 min. PCR products were purified and sequenced as described above. In order to exclude that the mutation c.1312C N T is a common polymorphism and to determine the actual allele frequency at this position, 100 alleles of healthy individuals were analyzed under the same conditions. A multiple sequence alignment of ALG1 and 13 homologous pseudogenes was made to verify that the primers ALG1-ex13F and ALG1-ex13R match the real ALG1 sequence only. For this purpose we used the Clustal Omega tool for multiple DNA sequence alignment under default settings. 2.6. Functional verification 2.6.1. Construction of the expression vector cDNA with the ALG1 coding sequence from a healthy control and the patient were amplified using the primers A1-10 F and A1-8RS (Kranz et al., 2004; Takahashi et al., 2000). They contain a recognition site for the restriction enzymes BamHI and SalI, which were used for cloning into the plasmid vector. For PCRamplification PCR Buffer 10× (Qiagen), A. dest., dNTPs (GE Healthcare), 2.5 μl DMSO and proof-reading polymerase Pfx (Invitrogen/Life technologies) were added to the cDNAs with the forward and reverse primer with a final reaction volume of 50μl. The samples were incubated at 95°C for 3min, followed by 35cycles of 95°C for 45s, 55°C for 45s, and 68 °C for 2.5 min and a final incubation at 72 °C for 10 min. Afterwards, both the purified PCR-products (QIAquick PCR Purification Kit, Qiagen) and the expression vector pYEX-BX (Clontech, Saint-Germain-en-Laye France, kindly provided by Dr. N. v. Deenen, Institute of Plant Biology and Biotechnology, Münster) were digested with BamHI and SalI (New England BioLabs, Frankfurt am Main, Germany). Samples were analyzed on 1% agarose gels and extracted with the QIAquick gel extraction kit (Qiagen). For cloning either the ALG1 wild-type sequence or each of the patient's point mutations c.1312C N T (R438W) and c.1145 T N C (M382T) into the vector pYEXBX, a Quick Ligation™ Kit (New England BioLabs) was used. After transformation and amplification in bacteria (TOP10F', Invitrogen/Life technologies), cloned vector constructs containing no insert, the wildtype sequence of ALG1 or one of the patient’s mutated ALG1 alleles were selected. To verify that the purified vector constructs carry the correct inserts, the coding region of all samples was sequenced and analyzed working with primers ALG1-IIF and ALG1-IIIR and the BigDye Terminator Kit 3.1 (Applied Biosystems/Life technologies). Samples cycled at 96 °C for 4 min and run for 35 cycles at 96 °C for 20 s, at 50 °C for 10 s and at 60 °C for 2 min and finally cooled down to 15 °C. Subsequently they were purified using Sephadex/Millipore System (GE Healthcare, Merck Millipore) and analyzed on an ABI PRISM 3730 sequencer. 2.6.2. Cell culture and yeast transformation The ALG1-deficient Saccharomyces cerevisiae strain PRY56 was used (kindly donated by Dr. C. Neupert, Prof. M. Aebi, Microbiology, ETH Zürich) for functional verification.
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Yeast cells were grown in YPD medium (1% yeast extract, 2% peptone, 2% glucose; pH 6.5) at 25 °C. Transformation of S. cerevisiae PRY56 was performed according to the freeze-method of Dohmen et al. (1991). Selection was done in YNB medium (Yeast nitrogen base, Difco, Detroit, USA) containing 2% glucose but no uracil. Functional complementation was done using the drop dilution assay essentially as described (Kranz et al., 2004) with the following modifications: 10-fold dilutions of yeast cells were dropped on YNB medium instead of YPD; induction of expression from pYEX-BX was achieved by adding 0.1 mM CuSO4 to the medium. Growth characteristics were compared after growing the cells at the permissive (26 °C) and the restrictive temperature (36 °C) for one week. 3. Results 3.1. 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 percentile). Mild dysmorphic features included multiple contractures at the finger joints (especially of fingers IV, V), at the large joints of the limbs and at his terminal joints. Arachnodactyly, broadened distal phalanges of the thumbs and clumsy, and partly crossed toes were present. An enlarged distance of the nipples, genital abnormalities like bilateral abdominal cryptorchidism and a marmorated pale skin were noted. Facial dysmorphism included micrognathia, abnormally shaped lowset ears, a large forehead, thin lips, a small mouth and small almondshaped eyes with a widened nasal bridge (Fig. 1). Ongoing feeding difficulties with a failure to thrive required partial parenteral nutrition. Nevertheless, within a few weeks growth retardation became obvious (body size 50 cm at 12 weeks, ~3rd percentile; see related growth parameters, Table 1) and secondary microcephaly developed. Recurrent non-infectious diarrhea was present and he had a profound hypoproteinemia. Renal protein loss was excluded. An increased amount of CDT in serum was found. Generalized oedema, massive ascites, pleural effusion and finally pericardial effusion developed. Symptomatic therapy with parenteral protein substitution and diuretics led to clinical stabilization. Seizures started at 3 months of age with single and grouped sharp wave patterns in electroencephalogram recordings. His seizures were
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Table 1 Abnormal laboratory parameters of patient JdL. Selected laboratory parameters Patient JdL
Reference range
Hypoproteinemia Total protein Serum albumin Transferrin Serum CDT
20 g/l 13 g/l 70 mg/dl 37.4%
65-80 g/l 35-52 g/l 202-302 mg/dl ≤2.6%
Immunoglobulins Immunoglobulin G
0.21 g/l
2.5-6.8 g/l
Growth parameters IGF1 IGFBP3
9 ng/ml 0.44 mg/l
60.4-217.4 ng/ml 2-5 mg/l
Heart parameters CK CK-MB CK-MB in % of CK Troponin T
375 U/l 94 U/l 25.07% 0.06 μg/l
≤171 U/l ≤11 U/l ≤6% ≤0.01 μg/l
Liver parameters ALT/GPT AST/GOT GLDH CHE
69 U/l 63 U/l 28 U/l 2.41 kU/l
≤45 U/l ≤35 U/l ≤7 U/l 5.32-12.92 kU/l
Coagulation parameters AT-III D-dimer Protein S, free
15% 4648 μg/l 47%
70-120% 0-500 μg/l 59-121%
Abbreviations: CDT = carbohydrate deficient transferrin, IGF1 = insulin-like growth factor 1, IGFBP3 = insulin-like growth factor-binding protein 3, CK = creatine kinase, CK-MB = creatine kinase-muscle-brain, ALT = alanine-aminotransferase/GPT = glutamic-pyruvic transaminase, AST = aspartate aminotransferase/GOT = glutamic oxaloacetic transaminase, GLDH = glutamate dehydrogenase, CHE = cholinesterase, AT-III = antithrombin-III.
refractory to anticonvulsive therapy with barbiturates (phenobarbital) and benzodiazepines (diazepam, clonazepam, midazolam). Episodes of prolonged apnoea occurred. Respiratory failure was treated by a nasal CPAP mask with oxygen supply and he received caffeine for respiratory stimulation. Hyperexcitability and muscular hypertension were present. Cardiac involvement was characterized by increased troponin and CK-MB levels (Table 1), an increased heart rate and paradoxical bradycardia upon stimulation. Echocardiography revealed no signs of cardiomyopathy. Abnormal laboratory parameters are listed in Table 1. Increased fibrinolysis products (D-dimer) were noted in the absence of thrombotic events. Agammoglobulinemia was present and also visualized by SDS-
Fig. 1. Clinical pictures of the ALG1-CDG patient JdL. Pictures of the affected boy show his facial dysmorphism with micrognathia (a), abnormally shaped low-set ears (a) and a widened nasal bridge (b).
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PAGE (Fig. 2). Repeatedly, increased inflammatory parameters (CRP 3.08–9 mg/dl) demanded antibiotic treatment. Recurrent seizures, episodes of prolonged apnoea and bradycardia finally resulted in cardiac arrest. After resuscitating the patient twice, the treatment was limited to palliative therapy and he died from cardiovascular failure at the age of 3 months and 23 days.
3.3. Analysis of lipid-linked oligosaccharides In order to investigate the assembly of Glc3Man9GlcNAc2 oligosaccharides, fibroblasts were labeled with [2-3H]-mannose and analyzed by HPLC. The patient's LLO pattern showed no structural LLO abnormalities but a reduction of the total LLO amount indicating an early defect in LLO biosynthesis before or at the incorporation of the first mannose into the LLO chain (data not shown).
3.2. CDG diagnostics 3.4. Analysis of the ALG1 gene and confirmation of R438W as disease causing Analyses of serum transferrin (IEF, HPLC, IMPP, SDS-PAGE). The common CDG screening test is the isoelectric focusing (IEF) of serum transferrin (Marquardt and Denecke, 2003). Serum transferrin has two branched oligosaccharide chains, each carrying two negatively charged sialic acid residues at the end (tetrasialotransferrin). In CDG-I patients an increased amount of disialo- and asialotransferrin occurs as the result of unoccupied glycosylation sites. In the patient, IEF revealed a CDG-I-pattern with normal patterns in the parents (Fig. 2). Quantification transferrin isoforms by HPLC revealed (reference values in brackets): 10.75% asialotransferrin (not detectable), 63.03% diasialotransferrin (1.10 ± 0.72), 3.08% trisialotransferrin (3.76 ± 2.60), 17.97% tetrasialotransferrin (89.94 ± 4.16) and 5.17% pentasialotransferrin (6.40 ± 3.80). Changes in the apparent molecular weight were illustrated by SDS-PAGE. SDS-PAGE of immunoprecipitated transferrin revealed the presence of two additional bands in the patient with an apparent molecular weight loss of 2 or 4 kDa, indicating the absence of one or both of the carbohydrate side chains (Fig. 2). Since IgG binds to protein A, the nearly complete absence of the IgG band in the patient's serum demonstrated his inability of synthesizing normal IgG-type immunoglobulins (see also Table 1).
Several genes encoding enzymes involved in the first steps of N-glycan biosynthesis were sequenced. Mutation analysis of the ALG1 gene revealed compound heterozygosity with two missense mutations: c.1145 T N C (M382T, paternal) in exon 11 and c.1312C N T (R438W, maternal) in exon 13 (Fig. 3). Recently published data (Morava et al., 2012) suggested that the mutation analysis of human ALG1 gene and its pseudogenes is prone to artifacts and that c.1312CNT is a common polymorphism. We analyzed 100 alleles of healthy donors without detection of c.1312C N T. We conclude that National Center for Biotechnology Information databank entry for the mutation c.1312C N T (R438W) in human ALG1 as a frequent polymorphism (rs16835020) has to be corrected. The entry was based on a CEPH control population of 184 chromosomes with a minor allele frequency of 0.140, corresponding to an average carrier frequency of 24% of the population, which we could not confirm. At the position corresponding to ALG1 c.1312 seven pseudogene sequences have a C as in wild-type ALG1, whereas five pseudogenes (ALG1L11P, ALG1L14P, ALG1L12P, ALG1L9P, ALG1L8P) have a T at this position. Multiple sequence alignment of ALG1 and its pseudogenes verified that the primers (ALG1-ex13F and ALG1-ex13R) only matched
Fig. 2. IEF and SDS-PAGE patterns of transferrin of a patient with CDG-Ia, a healthy control, patient JdL and his parents. The patient's IEF pattern shows an increased amount of asialo- and disialotransferrin, whereas the amount of full length oligosaccharides (tetrasialotransferrin) is decreased, characterizing a severe hypoglycosylation. The parent's IEF patterns are similar to the control. The patient's SDS-PAGE pattern shows two additional bands at the position of asialo- and disialotransferrin indicating the absence of one or two complete carbohydrate side chains in comparison to normally glycosylated transferrin as seen in the parents' and control's SDS-PAGE. In addition, a reduced amount of immunoglobulin IgG occurs in the patient's serum.
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Fig. 3. Mutation analysis gDNA of exon 11 and exon 13. Mutation analysis of the patient JdL and his parents: The patient's and the father's gDNA sequences carry the heterozygous ALG1 mutation c.1145 T N C, whereas the mother's ALG1 sequence is the same as the wild-type region of exon 11 (homozygous c.1145 T). A common single nucleotide polymorphism (rs1047732) can be detected in the patient's gDNA exon 11: c. 1149 C N T, which does not lead to an amino acid substitution (F383F). The patient's and the mother's gDNA sequences carry the heterozygous ALG1 mutation c.1312C N T, whereas the father's ALG1 sequence is the same as the wild-type region of exon 13 (homozygous c.1312 C).
the real ALG1 sequence excluding the accidental amplification of pseudogenes (Fig. 4).
growth similar to the mock transformed cells (Fig. 5), demonstrating the severely reduced function of both corresponding alleles.
3.5. Functional complementation of ALG1-deficient yeast strain PRY56 by transformation with the human ALG1-coding sequence
4. Discussion
ALG1-deficient yeast cells (PRY 56) with a temperature sensitive phenotype (Huffaker and Robbins, 1982) can be used to detect the functional consequences of mutated ALG1 alleles, since Takahashi et al. (2000) discovered that a transformation with the human ALG1 coding sequence complements the yeast's glycosylation and growth phenotype. To prove that the mutations c.1312C N T (R438W) and c.1145 T N C (M382T) do not correct impaired growth of PRY56, the yeast cell were transformed with the expression vector pYEX-BX (Kranz et al., 2004). The vector contained either no insert (mock), one of the patient's mutated alleles c.1312C N T (R438W) or c.1145 T N C (M382T) or the human wild-type ALG1 coding sequence. At the permissive temperature of 26 °C, growth characteristics of ALG1-deficient yeast cells containing the wild-type sequence were similar to those transformed with one of the mutated alleles or carrying no insert (mock). At the restrictive temperature of 36 °C, the human wild-type allele was capable to complement the temperature sensitive PRY 56 yeast cells whereas either one of the mutated alleles retained an impaired
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 dolichollinked oligosaccharides required for N-glycosylation on the cytosolic side of the ER membrane (Haeuptle and Hennet, 2009) and catalyzes the transfer of mannose onto GlcNAc2-PP-dolichol. First reports on ALG1-CDG patients in 2004 (Grubenmann et al., 2004; Kranz et al., 2004; Schwarz et al., 2004) described a severe phenotype leading to death in the first months of life. Recently, patients with a milder phenotype were reported (Dupré et al., 2010; Morava et al., 2012). The present case had a particularly severe phenotype that resulted in early death. He showed feeding problems and diarrhea, hypoproteinemia, oedema, seizures, cardiac and hepatic involvement, coagulation anomalies and dysmorphic features. An especially severe hypoglycosylation with a profound reduction of tetrasialotransferrin was present.
Fig. 4. Multiple sequence alignment of ALG1 and its pseudogenes in the section from ALG1-ex13F to ALG1-ex13R. The alignment shows deviations between ALG1 and its pseudogenes (highlighted in pink) regarding the primer sequences (highlighted in yellow), the section between these primers and the mutation site c.1312 (highlighted in green). Ensembl-IDs of the pseudogenes are given in supplementary Table 2.
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Fig. 5. Functional complementation of the temperature sensitive ALG1-1 mutant S. cerevisiae PRY56. Yeast cells were transformed with the expression vector containing the wild-type coding region of ALG1, either one of the mutated alleles (1312C N T; 1145 T N C), or the empty vector pYEX-BX (mock). Sequential 10-fold dilutions were dropped on YNB agar plates with 0.1 mM CuSO4 for induction. Growth characteristics were compared either at the permissive (26 °C) or at the restrictive temperature (36 °C) after incubation for one week, showing ALG1-deficient PRY 56 yeast cells could be complemented by transformation with the wild-type coding region of human ALG1, whereas the yeast's growth with each of the patient's mutated ALG1 sequences is decreased.
Agammaglobulinaemia, as observed in our patient, was present in only one of the previously published ALG1-CDG patients (Kranz et al., 2004; Morava et al., 2012). In contrast to the other ALG1-CDG cases, our patient did not present any ocular manifestations (de Koning et al., 1998; Dupré et al., 2010; Grubenmann et al., 2004; Kranz et al., 2004; Morava et al., 2012). Furthermore, the patient suffered from generalized muscular hypertonia in contrast to the occurrence of hypotonia, as reported in the majority of ALG1-CDG patients (Dupré et al., 2010; Grubenmann et al., 2004; Kranz et al., 2004; Morava et al., 2012). Up to now only one sib pair has been reported having multiple contractures as our case. Both showed a severe course and died within first two weeks of life (de Koning et al., 1998). Both mutations were previously described in two other patients (Dupré et al., 2010; Morava et al., 2012). By functional analysis of these mutations in yeast cells we could prove their pathogenicity. Multiple sequence alignments of ALG1 with homologous proteins from different species show domains of strong sequence conservation at the C-terminus of the protein, emphasizing the assumption that the catalytically active site of the enzyme is located at this region (Grubenmann et al., 2004; Morava et al., 2012). In yeast, the C-terminal region of the ALG1 protein has physical interactions with other mannosyltransferases (ALG1, ALG2 and ALG11) (Gao et al., 2004). Both amino acid exchanges found in our patient are located within this conserved cytosolic C-terminal domain of the mannosyltransferase. The first mutation, M382T, was recently published in combination with the heterozygous mutation c.893C N T (A298V) (Morava et al., 2012). The patient II/3 had a milder phenotype. Since we have shown that M382T has no relevant residual function, A298V must be a milder mutation. The second mutation, R438W, has been described in another patient P1 in combination with a loss of function mutation on the other allele (Dupré et al., 2010). As in our patient, death occurred in the first year of life indicating that the R438W mutation is severe. R438W was previously described as a common polymorphism with a minor allele frequency of 0.140 and a predicted carrier frequency of 24% (Morava et al., 2012). However, there are two cases of ALG1-CDG patients with this amino acid substitution, one was reported in 2010 (Dupré et al., 2010) and the other one is our patient. In both cases, a severely reduced function of the enzyme was shown. Combining both studies, 132 unrelated healthy controls (264 alleles) were sequenced without finding any mutated allele (Dupré et al., 2010). This may be due to the fact that the mutation analysis of the human ALG1 gene is prone to artifacts due to the existence of several pseudogenes. A multiple sequence alignment of ALG1 and its pseudogenes in the section from ALG1-ex13F to ALG1-
ex13R depicting several deviations in the sequence context confirms the exclusive amplification of the human ALG1 gene (Fig. 4). The accidental amplification of particularly those five pseudogenes, which actually contain a T at the position corresponding to ALG1 c.1312, could thus be excluded by the present examination. The accidental amplification of these pseudogenes might lead to the assumption that the mutation c.1312C N T (R438W) could be a frequent polymorphism (rs16835020) as listed in the National Center for Biotechnology Information database. These data were based on examinations of 92 unrelated individuals of a CEPH control population using the sequenom technology, where the mutation site is sequenced in a one base extension only, not revealing the sequence context, and do neither accord with the results of previous studies (Dupré et al., 2010) nor with the present investigations. Thus, the statement that the high frequency of SNP rs16835020 (0.140) listed in databases is due to the pseudogenes (Morava et al., 2012) likely is correct and thus the SNPentry is wrong. However, the conclusion that the c.1312C N T mutation in ALG1 is also due to amplification of the pseudogenes is incorrect as demonstrated here with ALG1-specific primers and therefore the mutation c.1312C N T (R438W) should be listed as a real diseasecausing mutation. ALG1-CDG has a wide spectrum of clinical appearances and still presents one of the most severe phenotypes in the growing group of congenital disorders of glycosylation. 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 pYEXBX (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. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2013.10.013. References Biffi, S., Tamaro, G., Bortot, B., Zamberlan, S., Severini, G.M., Carrozzi, M., 2007. Carbohydrate-deficient transferrin (CDT) as a biochemical tool for the screening of congenital disorders of glycosylation (CDGs). Clin. Biochem. 40, 1431–1434. Chantret, I., et al., 2003. A deficiency in dolichyl-P-glucose:Glc1Man9GlcNAc2-PP-dolichyl alpha3-glucosyltransferase defines a new subtype of congenital disorders of glycosylation. J. Biol. Chem. 278, 9962–9971. Couto, J.R., Huffaker, T.C., Robbins, P.W., 1984. Cloning and expression in Escherichia coli of a yeast mannosyltransferase from the asparagine-linked glycosylation pathway. J. Biol. Chem. 259, 378–382. de Koning, T.J., et al., 1998. Recurrent nonimmune hydrops fetalis associated with carbohydrate-deficient glycoprotein syndrome. J. Inherit. Metab. Dis. 21, 681–682. Dohmen, R.J., Strasser, A.W., Höner, C.B., Hollenberg, C.P., 1991. An efficient transformation procedure enabling long-term storage of competent cells of various yeast genera. Yeast 7, 691–692. Dupré, T., et al., 2010. Guanosine diphosphate-mannose:GlcNAc2-PP-dolichol mannosyltransferase deficiency (congenital disorders of glycosylation type Ik): five new patients and seven novel mutations. J. Med. Genet. 47, 729–735. Gao, X.D., Nishikawa, A., Dean, N., 2004. Physical interactions between the Alg1, Alg2, and Alg11 mannosyltransferases of the endoplasmic reticulum. Glycobiology 14, 559–570. Grubenmann, C.E., et al., 2004. Deficiency of the first mannosylation step in the N-glycosylation pathway causes congenital disorder of glycosylation type Ik. Hum. Mol. Genet. 13, 535–542.
A.-K. Rohlfing et al. / Gene 534 (2014) 345–351 Haeuptle, M.A., Hennet, T., 2009. Congenital disorders of glycosylation: an update on defects affecting the biosynthesis of dolichol-linked oligosaccharides. Hum. Mutat. 30, 1628–1641. Huffaker, T.C., Robbins, P.W., 1982. Temperature-sensitive yeast mutants deficient in asparagine-linked glycosylation. J. Biol. Chem. 257, 3203–3210. Jaeken, J., 2010. Congenital disorders of glycosylation. Ann. N. Y. Acad. Sci. 1214, 190–198. Jaeken, J., Vanderschueren-Lodeweyckx, M., Casaer, P., 1980. Familial psychomotor retardation with markedly fluctuating serum prolactin, FSH and GH levels, partial TBG deficiency, increased serum arylsulfatase A and increased CSF protein: a new syndrome? Pediatr. Res. 14, 17. Kranz, C., et al., 2001. A mutation in the human MPDU1 gene causes congenital disorder of glycosylation type If (CDG-If). J. Clin. Invest. 108, 1613–1619. Kranz, C., et al., 2004. Congenital disorder of glycosylation type Ik (CDG-Ik): a defect of mannosyltransferase I. Am. J. Hum. Genet. 74, 545–551. Marquardt, T., Denecke, J., 2003. Congenital disorders of glycosylation: review of their molecular bases, clinical presentations and specific therapies. Eur. J. Pediatr. 162, 359–379.
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Morava, E., et al., 2012. Defining the phenotype in congenital disorder of glycosylation due to ALG1 mutations. Pediatrics 130, e1034–e1039. Niehues, R., et al., 1998. Carbohydrate-deficient glycoprotein syndrome type Ib. Phosphomannose isomerase deficiency and mannose therapy. J. Clin. Invest. 101, 1414–1420. Schwarz, M., et al., 2004. Deficiency of GDP-Man:GlcNAc2-PP-dolichol mannosyltransferase causes congenital disorder of glycosylation type Ik. Am. J. Hum. Genet. 74, 472–481. Snow, T.M., Woods, C.W., Woods, A.G., 2012. Congenital disorder of glycosylation: a case presentation. Adv. Neonatal Care 12, 96–100. Takahashi, T., Honda, R., Nishikawa, Y., 2000. Cloning of the human cDNA which can complement the defect of the yeast mannosyltransferase I-deficient mutant alg 1. Glycobiology 10, 321–327. Theodore, M., Morava, E., 2011. Congenital disorders of glycosylation: sweet news. Curr. Opin. Pediatr. 23, 581–587. Würde, A.E., et al., 2012. Congenital disorder of glycosylation type Ij (CDG-Ij, DPAGT1CDG): extending the clinical and molecular spectrum of a rare disease. Mol. Genet. Metab. 105, 634–641.
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Gene journal homepage: www.elsevier.com/locate/gene
ALG1-CDG: A new case with early fatal outcome A.-K. Rohlfing a,1, S. Rust b, J. Reunert a,1, M. Tirre b, I. Du Chesne a,1, Sa. Wemhoff c, F. Meinhardt c, H. Hartmann d, A.M. Das d, T. Marquardt a,⁎,1 a
Universitätsklinikum Münster, Klinik und Poliklinik für Kinder- und Jugendmedizin-Allgemeine Pädiatrie, Münster, Germany Leibniz-Institut für Arterioskleroseforschung, Münster, Germany Institut für Molekulare Mikrobiologie und Biotechnologie, Münster, Germany d Clinic for Pediatric Kidney, Liver Metabolic Diseases, Hannover Medical School, Hannover, Germany b c
a r t i c l e
i n f o
Article history: Accepted 8 October 2013 Available online 21 October 2013 Keywords: Congenital disorder of glycosylation type Ik Hypoglycosylation ALG1 pseudogenes Seizures Hypoproteinemia
a b s t r a c t 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 N C (M382T) and c.1312C N 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. © 2013 Elsevier B.V. All rights reserved.
1. 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 Abbreviations: ALG1, Asparagine-linked glycosylation 1 homolog (yeast, beta-1,4mannosyltransferase) = chitobiosyldiphosphodolichol beta-mannosyltransferase; CDG, Congenital disorders of glycosylation; GDP, Guanosine diphosphate; Man, Mannose; RER, Rough endoplasmic reticulum; HMT1, Human mannosyltransferase 1; GlcNAc, N-acetyl-D-glucosamine; PP, Pyrophosphate; kDa, Kilodaltons; CDT, Carbohydrate deficient transferrin; IEF, Isoelectric focusing; IMPP, Immunoprecipitation; SDS, Sodium dodecyl sulfate; PAGE, Polyacrylamide-gel electrophoresis; HPLC, High performance liquid chromatography; MEM, Minimum essential medium; PBS, Phosphate buffered saline; LLO, Lipid-linked oligosaccharides; DMEM, Dulbecco's Modified Eagle's Medium; cDNA, DNA complementary to RNA; EDTA, Ethylenediaminetetraacetic acid; PCR, Polymerase chain reaction; dNTP, Deoxyribonucleoside triphosphate; DMSO, Demethylsulfoxide; CPAP, Continuous positive airway pressure; CK, Creatine kinase; CK-MB, Creatine kinase muscle-brain, i.e. myocardial subtype of CK; CRP, C-reactive protein (reacting with C-polysaccharide of Pneumococcus); IgG, Immunoglobulin G; Glc, Glucose; CEPH, Centre d'Etude du Polymorphisme Humain; DPAGT1, Dolichyl-phosphate (UDP-N-acetylglucosamine) N-acetylglucosaminephosphotransferase 1; IGF1, Insulin-like growth factor 1; IGFBP3, Insulin-like growth factor-binding protein 3; ALT, Alanine-aminotransferase; GPT, Glutamic-pyruvic transaminase; AST, Aspartate aminotransferase; GOT, Glutamic oxaloacetic transaminase; GLDH, Glutamate dehydrogenase; CHE, Cholinesterase; AT-III, Antithrombin-III. ⁎ Corresponding author at: Department of Pediatrics University Hospital of Münster Albert-Schweitzer-Campus 1 48149 Münster, Germany. E-mail address: [email protected] (T. Marquardt). 1 Tel.: +49 251 8358519; fax: +49 251 8356085. 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.10.013
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-PPdolichol mannosyltransferase (alias chitobiosyldiphosphodolichol betamannosyltransferase; 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
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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 ALG1CDG. 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. 2. Materials and methods 2.1. 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). 2.2. 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 10% fetal bovine serum (Invitrogen/Life Technologies, Darmstadt, Germany), 2mM L-glutamine, 100 μg/ml penicillin and streptomycin. For subsequent RNA- and DNAextractions fibroblasts were obtained by trypsin treatment and washed in phosphate buffered saline (PBS) (PAA) before they were resuspended in 200 μl 0.9% NaCl and stored at −80 °C (Würde et al., 2012). 2.3. LLO analysis Fibroblasts from the patient and control cells were labeled with 100 μCi [2-3H]-mannose per ml labeling medium (DMEM without glucose: MEM, 9:1) for 45 min, incubated in MEM for 10 min, and subsequently washed with PBS as described previously (Chantret et al., 2003). The metabolically labeled dolicholpyrophosphate-linked oligosaccharides were extracted, purified and analyzed using high performance liquid chromatography (HPLC) as described (Kranz et al., 2001). 2.4. Mutation analysis RNA was isolated from control and patient fibroblasts by using the Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany). For generating cDNA, RNA was transcribed with SuperScript™ III Reverse Transcriptase (Invitrogen/Life technologies) and oligo (dT) primers (Fermentas, St. Leon-Rot, Germany). EDTA blood samples were taken from the patient and his parents to isolate genomic DNA with QIAmp DNA mini kit (Qiagen). The ALG1 coding sequence (NM_019109.4) was amplified with specific primers and PCR conditions (Supplementary Table 1). The amplified PCR products were purified with the PCR Product PreSequencing Kit (USB Products/Affymetrix, Ohio, USA). Sequencing was performed using BigDye Terminator Kit 3.1 (Applied Biosystems/Life technologies, Darmstadt, Germany) and the primers used for PCR under the following conditions: 96 °C for 2 min followed by 25 cycles at 94°C for 10s, at 50°C for 5s and at 60°C for 2min. Samples were purified using Sephadex/Millipore System (GE Healthcare, Buckinghamshire, UK/Merck Millipore, Schwalbach, Germany), and analyzed on an ABI PRISM 3730 sequencer. For confirmation of the patient’s ALG1 mutations at the genomic level, exon 11 and 13 were amplified (primers and PCR conditions are listed in Supplementary Table 1). Specific primers were designed to differentiate between the ALG1 gene (NG_009202.1) and its documented pseudogenes. PCR products were purified and sequenced as described above using universal primers (Supplementary Table 1).
2.5. Investigation of the mutation c.1312C N T in exon 13 In order to make sure that the heterozygous mutation c.1312C N T in the patient’s DNA was not only an artifact due to amplification of pseudogene sequences homologous to ALG1, as suggested in (Morava et al., 2012), the genomic ALG1 sequences of the patient and a healthy control were amplified under more stringent conditions using the HotStarTaq DNA Polymerase (Qiagen) and primers ALG1-ex13F and ALG1-ex13R. The Master Mix also contained 10× PCR Buffer (Qiagen), 5× Q-Solution (Qiagen), MgCl2 (Qiagen), A. dest. and dNTPs (GE Healthcare). For PCR amplification, samples were incubated at 94 °C for 15 min followed by 35 cycles of 94 °C for 30 s, 65 °C for 30 s, and 72 °C for 30 s, and a final incubation at 72 °C for 7 min. PCR products were purified and sequenced as described above. In order to exclude that the mutation c.1312C N T is a common polymorphism and to determine the actual allele frequency at this position, 100 alleles of healthy individuals were analyzed under the same conditions. A multiple sequence alignment of ALG1 and 13 homologous pseudogenes was made to verify that the primers ALG1-ex13F and ALG1-ex13R match the real ALG1 sequence only. For this purpose we used the Clustal Omega tool for multiple DNA sequence alignment under default settings. 2.6. Functional verification 2.6.1. Construction of the expression vector cDNA with the ALG1 coding sequence from a healthy control and the patient were amplified using the primers A1-10 F and A1-8RS (Kranz et al., 2004; Takahashi et al., 2000). They contain a recognition site for the restriction enzymes BamHI and SalI, which were used for cloning into the plasmid vector. For PCRamplification PCR Buffer 10× (Qiagen), A. dest., dNTPs (GE Healthcare), 2.5 μl DMSO and proof-reading polymerase Pfx (Invitrogen/Life technologies) were added to the cDNAs with the forward and reverse primer with a final reaction volume of 50μl. The samples were incubated at 95°C for 3min, followed by 35cycles of 95°C for 45s, 55°C for 45s, and 68 °C for 2.5 min and a final incubation at 72 °C for 10 min. Afterwards, both the purified PCR-products (QIAquick PCR Purification Kit, Qiagen) and the expression vector pYEX-BX (Clontech, Saint-Germain-en-Laye France, kindly provided by Dr. N. v. Deenen, Institute of Plant Biology and Biotechnology, Münster) were digested with BamHI and SalI (New England BioLabs, Frankfurt am Main, Germany). Samples were analyzed on 1% agarose gels and extracted with the QIAquick gel extraction kit (Qiagen). For cloning either the ALG1 wild-type sequence or each of the patient's point mutations c.1312C N T (R438W) and c.1145 T N C (M382T) into the vector pYEXBX, a Quick Ligation™ Kit (New England BioLabs) was used. After transformation and amplification in bacteria (TOP10F', Invitrogen/Life technologies), cloned vector constructs containing no insert, the wildtype sequence of ALG1 or one of the patient’s mutated ALG1 alleles were selected. To verify that the purified vector constructs carry the correct inserts, the coding region of all samples was sequenced and analyzed working with primers ALG1-IIF and ALG1-IIIR and the BigDye Terminator Kit 3.1 (Applied Biosystems/Life technologies). Samples cycled at 96 °C for 4 min and run for 35 cycles at 96 °C for 20 s, at 50 °C for 10 s and at 60 °C for 2 min and finally cooled down to 15 °C. Subsequently they were purified using Sephadex/Millipore System (GE Healthcare, Merck Millipore) and analyzed on an ABI PRISM 3730 sequencer. 2.6.2. Cell culture and yeast transformation The ALG1-deficient Saccharomyces cerevisiae strain PRY56 was used (kindly donated by Dr. C. Neupert, Prof. M. Aebi, Microbiology, ETH Zürich) for functional verification.
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Yeast cells were grown in YPD medium (1% yeast extract, 2% peptone, 2% glucose; pH 6.5) at 25 °C. Transformation of S. cerevisiae PRY56 was performed according to the freeze-method of Dohmen et al. (1991). Selection was done in YNB medium (Yeast nitrogen base, Difco, Detroit, USA) containing 2% glucose but no uracil. Functional complementation was done using the drop dilution assay essentially as described (Kranz et al., 2004) with the following modifications: 10-fold dilutions of yeast cells were dropped on YNB medium instead of YPD; induction of expression from pYEX-BX was achieved by adding 0.1 mM CuSO4 to the medium. Growth characteristics were compared after growing the cells at the permissive (26 °C) and the restrictive temperature (36 °C) for one week. 3. Results 3.1. 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 percentile). Mild dysmorphic features included multiple contractures at the finger joints (especially of fingers IV, V), at the large joints of the limbs and at his terminal joints. Arachnodactyly, broadened distal phalanges of the thumbs and clumsy, and partly crossed toes were present. An enlarged distance of the nipples, genital abnormalities like bilateral abdominal cryptorchidism and a marmorated pale skin were noted. Facial dysmorphism included micrognathia, abnormally shaped lowset ears, a large forehead, thin lips, a small mouth and small almondshaped eyes with a widened nasal bridge (Fig. 1). Ongoing feeding difficulties with a failure to thrive required partial parenteral nutrition. Nevertheless, within a few weeks growth retardation became obvious (body size 50 cm at 12 weeks, ~3rd percentile; see related growth parameters, Table 1) and secondary microcephaly developed. Recurrent non-infectious diarrhea was present and he had a profound hypoproteinemia. Renal protein loss was excluded. An increased amount of CDT in serum was found. Generalized oedema, massive ascites, pleural effusion and finally pericardial effusion developed. Symptomatic therapy with parenteral protein substitution and diuretics led to clinical stabilization. Seizures started at 3 months of age with single and grouped sharp wave patterns in electroencephalogram recordings. His seizures were
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Table 1 Abnormal laboratory parameters of patient JdL. Selected laboratory parameters Patient JdL
Reference range
Hypoproteinemia Total protein Serum albumin Transferrin Serum CDT
20 g/l 13 g/l 70 mg/dl 37.4%
65-80 g/l 35-52 g/l 202-302 mg/dl ≤2.6%
Immunoglobulins Immunoglobulin G
0.21 g/l
2.5-6.8 g/l
Growth parameters IGF1 IGFBP3
9 ng/ml 0.44 mg/l
60.4-217.4 ng/ml 2-5 mg/l
Heart parameters CK CK-MB CK-MB in % of CK Troponin T
375 U/l 94 U/l 25.07% 0.06 μg/l
≤171 U/l ≤11 U/l ≤6% ≤0.01 μg/l
Liver parameters ALT/GPT AST/GOT GLDH CHE
69 U/l 63 U/l 28 U/l 2.41 kU/l
≤45 U/l ≤35 U/l ≤7 U/l 5.32-12.92 kU/l
Coagulation parameters AT-III D-dimer Protein S, free
15% 4648 μg/l 47%
70-120% 0-500 μg/l 59-121%
Abbreviations: CDT = carbohydrate deficient transferrin, IGF1 = insulin-like growth factor 1, IGFBP3 = insulin-like growth factor-binding protein 3, CK = creatine kinase, CK-MB = creatine kinase-muscle-brain, ALT = alanine-aminotransferase/GPT = glutamic-pyruvic transaminase, AST = aspartate aminotransferase/GOT = glutamic oxaloacetic transaminase, GLDH = glutamate dehydrogenase, CHE = cholinesterase, AT-III = antithrombin-III.
refractory to anticonvulsive therapy with barbiturates (phenobarbital) and benzodiazepines (diazepam, clonazepam, midazolam). Episodes of prolonged apnoea occurred. Respiratory failure was treated by a nasal CPAP mask with oxygen supply and he received caffeine for respiratory stimulation. Hyperexcitability and muscular hypertension were present. Cardiac involvement was characterized by increased troponin and CK-MB levels (Table 1), an increased heart rate and paradoxical bradycardia upon stimulation. Echocardiography revealed no signs of cardiomyopathy. Abnormal laboratory parameters are listed in Table 1. Increased fibrinolysis products (D-dimer) were noted in the absence of thrombotic events. Agammoglobulinemia was present and also visualized by SDS-
Fig. 1. Clinical pictures of the ALG1-CDG patient JdL. Pictures of the affected boy show his facial dysmorphism with micrognathia (a), abnormally shaped low-set ears (a) and a widened nasal bridge (b).
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PAGE (Fig. 2). Repeatedly, increased inflammatory parameters (CRP 3.08–9 mg/dl) demanded antibiotic treatment. Recurrent seizures, episodes of prolonged apnoea and bradycardia finally resulted in cardiac arrest. After resuscitating the patient twice, the treatment was limited to palliative therapy and he died from cardiovascular failure at the age of 3 months and 23 days.
3.3. Analysis of lipid-linked oligosaccharides In order to investigate the assembly of Glc3Man9GlcNAc2 oligosaccharides, fibroblasts were labeled with [2-3H]-mannose and analyzed by HPLC. The patient's LLO pattern showed no structural LLO abnormalities but a reduction of the total LLO amount indicating an early defect in LLO biosynthesis before or at the incorporation of the first mannose into the LLO chain (data not shown).
3.2. CDG diagnostics 3.4. Analysis of the ALG1 gene and confirmation of R438W as disease causing Analyses of serum transferrin (IEF, HPLC, IMPP, SDS-PAGE). The common CDG screening test is the isoelectric focusing (IEF) of serum transferrin (Marquardt and Denecke, 2003). Serum transferrin has two branched oligosaccharide chains, each carrying two negatively charged sialic acid residues at the end (tetrasialotransferrin). In CDG-I patients an increased amount of disialo- and asialotransferrin occurs as the result of unoccupied glycosylation sites. In the patient, IEF revealed a CDG-I-pattern with normal patterns in the parents (Fig. 2). Quantification transferrin isoforms by HPLC revealed (reference values in brackets): 10.75% asialotransferrin (not detectable), 63.03% diasialotransferrin (1.10 ± 0.72), 3.08% trisialotransferrin (3.76 ± 2.60), 17.97% tetrasialotransferrin (89.94 ± 4.16) and 5.17% pentasialotransferrin (6.40 ± 3.80). Changes in the apparent molecular weight were illustrated by SDS-PAGE. SDS-PAGE of immunoprecipitated transferrin revealed the presence of two additional bands in the patient with an apparent molecular weight loss of 2 or 4 kDa, indicating the absence of one or both of the carbohydrate side chains (Fig. 2). Since IgG binds to protein A, the nearly complete absence of the IgG band in the patient's serum demonstrated his inability of synthesizing normal IgG-type immunoglobulins (see also Table 1).
Several genes encoding enzymes involved in the first steps of N-glycan biosynthesis were sequenced. Mutation analysis of the ALG1 gene revealed compound heterozygosity with two missense mutations: c.1145 T N C (M382T, paternal) in exon 11 and c.1312C N T (R438W, maternal) in exon 13 (Fig. 3). Recently published data (Morava et al., 2012) suggested that the mutation analysis of human ALG1 gene and its pseudogenes is prone to artifacts and that c.1312CNT is a common polymorphism. We analyzed 100 alleles of healthy donors without detection of c.1312C N T. We conclude that National Center for Biotechnology Information databank entry for the mutation c.1312C N T (R438W) in human ALG1 as a frequent polymorphism (rs16835020) has to be corrected. The entry was based on a CEPH control population of 184 chromosomes with a minor allele frequency of 0.140, corresponding to an average carrier frequency of 24% of the population, which we could not confirm. At the position corresponding to ALG1 c.1312 seven pseudogene sequences have a C as in wild-type ALG1, whereas five pseudogenes (ALG1L11P, ALG1L14P, ALG1L12P, ALG1L9P, ALG1L8P) have a T at this position. Multiple sequence alignment of ALG1 and its pseudogenes verified that the primers (ALG1-ex13F and ALG1-ex13R) only matched
Fig. 2. IEF and SDS-PAGE patterns of transferrin of a patient with CDG-Ia, a healthy control, patient JdL and his parents. The patient's IEF pattern shows an increased amount of asialo- and disialotransferrin, whereas the amount of full length oligosaccharides (tetrasialotransferrin) is decreased, characterizing a severe hypoglycosylation. The parent's IEF patterns are similar to the control. The patient's SDS-PAGE pattern shows two additional bands at the position of asialo- and disialotransferrin indicating the absence of one or two complete carbohydrate side chains in comparison to normally glycosylated transferrin as seen in the parents' and control's SDS-PAGE. In addition, a reduced amount of immunoglobulin IgG occurs in the patient's serum.
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Fig. 3. Mutation analysis gDNA of exon 11 and exon 13. Mutation analysis of the patient JdL and his parents: The patient's and the father's gDNA sequences carry the heterozygous ALG1 mutation c.1145 T N C, whereas the mother's ALG1 sequence is the same as the wild-type region of exon 11 (homozygous c.1145 T). A common single nucleotide polymorphism (rs1047732) can be detected in the patient's gDNA exon 11: c. 1149 C N T, which does not lead to an amino acid substitution (F383F). The patient's and the mother's gDNA sequences carry the heterozygous ALG1 mutation c.1312C N T, whereas the father's ALG1 sequence is the same as the wild-type region of exon 13 (homozygous c.1312 C).
the real ALG1 sequence excluding the accidental amplification of pseudogenes (Fig. 4).
growth similar to the mock transformed cells (Fig. 5), demonstrating the severely reduced function of both corresponding alleles.
3.5. Functional complementation of ALG1-deficient yeast strain PRY56 by transformation with the human ALG1-coding sequence
4. Discussion
ALG1-deficient yeast cells (PRY 56) with a temperature sensitive phenotype (Huffaker and Robbins, 1982) can be used to detect the functional consequences of mutated ALG1 alleles, since Takahashi et al. (2000) discovered that a transformation with the human ALG1 coding sequence complements the yeast's glycosylation and growth phenotype. To prove that the mutations c.1312C N T (R438W) and c.1145 T N C (M382T) do not correct impaired growth of PRY56, the yeast cell were transformed with the expression vector pYEX-BX (Kranz et al., 2004). The vector contained either no insert (mock), one of the patient's mutated alleles c.1312C N T (R438W) or c.1145 T N C (M382T) or the human wild-type ALG1 coding sequence. At the permissive temperature of 26 °C, growth characteristics of ALG1-deficient yeast cells containing the wild-type sequence were similar to those transformed with one of the mutated alleles or carrying no insert (mock). At the restrictive temperature of 36 °C, the human wild-type allele was capable to complement the temperature sensitive PRY 56 yeast cells whereas either one of the mutated alleles retained an impaired
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 dolichollinked oligosaccharides required for N-glycosylation on the cytosolic side of the ER membrane (Haeuptle and Hennet, 2009) and catalyzes the transfer of mannose onto GlcNAc2-PP-dolichol. First reports on ALG1-CDG patients in 2004 (Grubenmann et al., 2004; Kranz et al., 2004; Schwarz et al., 2004) described a severe phenotype leading to death in the first months of life. Recently, patients with a milder phenotype were reported (Dupré et al., 2010; Morava et al., 2012). The present case had a particularly severe phenotype that resulted in early death. He showed feeding problems and diarrhea, hypoproteinemia, oedema, seizures, cardiac and hepatic involvement, coagulation anomalies and dysmorphic features. An especially severe hypoglycosylation with a profound reduction of tetrasialotransferrin was present.
Fig. 4. Multiple sequence alignment of ALG1 and its pseudogenes in the section from ALG1-ex13F to ALG1-ex13R. The alignment shows deviations between ALG1 and its pseudogenes (highlighted in pink) regarding the primer sequences (highlighted in yellow), the section between these primers and the mutation site c.1312 (highlighted in green). Ensembl-IDs of the pseudogenes are given in supplementary Table 2.
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Fig. 5. Functional complementation of the temperature sensitive ALG1-1 mutant S. cerevisiae PRY56. Yeast cells were transformed with the expression vector containing the wild-type coding region of ALG1, either one of the mutated alleles (1312C N T; 1145 T N C), or the empty vector pYEX-BX (mock). Sequential 10-fold dilutions were dropped on YNB agar plates with 0.1 mM CuSO4 for induction. Growth characteristics were compared either at the permissive (26 °C) or at the restrictive temperature (36 °C) after incubation for one week, showing ALG1-deficient PRY 56 yeast cells could be complemented by transformation with the wild-type coding region of human ALG1, whereas the yeast's growth with each of the patient's mutated ALG1 sequences is decreased.
Agammaglobulinaemia, as observed in our patient, was present in only one of the previously published ALG1-CDG patients (Kranz et al., 2004; Morava et al., 2012). In contrast to the other ALG1-CDG cases, our patient did not present any ocular manifestations (de Koning et al., 1998; Dupré et al., 2010; Grubenmann et al., 2004; Kranz et al., 2004; Morava et al., 2012). Furthermore, the patient suffered from generalized muscular hypertonia in contrast to the occurrence of hypotonia, as reported in the majority of ALG1-CDG patients (Dupré et al., 2010; Grubenmann et al., 2004; Kranz et al., 2004; Morava et al., 2012). Up to now only one sib pair has been reported having multiple contractures as our case. Both showed a severe course and died within first two weeks of life (de Koning et al., 1998). Both mutations were previously described in two other patients (Dupré et al., 2010; Morava et al., 2012). By functional analysis of these mutations in yeast cells we could prove their pathogenicity. Multiple sequence alignments of ALG1 with homologous proteins from different species show domains of strong sequence conservation at the C-terminus of the protein, emphasizing the assumption that the catalytically active site of the enzyme is located at this region (Grubenmann et al., 2004; Morava et al., 2012). In yeast, the C-terminal region of the ALG1 protein has physical interactions with other mannosyltransferases (ALG1, ALG2 and ALG11) (Gao et al., 2004). Both amino acid exchanges found in our patient are located within this conserved cytosolic C-terminal domain of the mannosyltransferase. The first mutation, M382T, was recently published in combination with the heterozygous mutation c.893C N T (A298V) (Morava et al., 2012). The patient II/3 had a milder phenotype. Since we have shown that M382T has no relevant residual function, A298V must be a milder mutation. The second mutation, R438W, has been described in another patient P1 in combination with a loss of function mutation on the other allele (Dupré et al., 2010). As in our patient, death occurred in the first year of life indicating that the R438W mutation is severe. R438W was previously described as a common polymorphism with a minor allele frequency of 0.140 and a predicted carrier frequency of 24% (Morava et al., 2012). However, there are two cases of ALG1-CDG patients with this amino acid substitution, one was reported in 2010 (Dupré et al., 2010) and the other one is our patient. In both cases, a severely reduced function of the enzyme was shown. Combining both studies, 132 unrelated healthy controls (264 alleles) were sequenced without finding any mutated allele (Dupré et al., 2010). This may be due to the fact that the mutation analysis of the human ALG1 gene is prone to artifacts due to the existence of several pseudogenes. A multiple sequence alignment of ALG1 and its pseudogenes in the section from ALG1-ex13F to ALG1-
ex13R depicting several deviations in the sequence context confirms the exclusive amplification of the human ALG1 gene (Fig. 4). The accidental amplification of particularly those five pseudogenes, which actually contain a T at the position corresponding to ALG1 c.1312, could thus be excluded by the present examination. The accidental amplification of these pseudogenes might lead to the assumption that the mutation c.1312C N T (R438W) could be a frequent polymorphism (rs16835020) as listed in the National Center for Biotechnology Information database. These data were based on examinations of 92 unrelated individuals of a CEPH control population using the sequenom technology, where the mutation site is sequenced in a one base extension only, not revealing the sequence context, and do neither accord with the results of previous studies (Dupré et al., 2010) nor with the present investigations. Thus, the statement that the high frequency of SNP rs16835020 (0.140) listed in databases is due to the pseudogenes (Morava et al., 2012) likely is correct and thus the SNPentry is wrong. However, the conclusion that the c.1312C N T mutation in ALG1 is also due to amplification of the pseudogenes is incorrect as demonstrated here with ALG1-specific primers and therefore the mutation c.1312C N T (R438W) should be listed as a real diseasecausing mutation. ALG1-CDG has a wide spectrum of clinical appearances and still presents one of the most severe phenotypes in the growing group of congenital disorders of glycosylation. 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 pYEXBX (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. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2013.10.013. References Biffi, S., Tamaro, G., Bortot, B., Zamberlan, S., Severini, G.M., Carrozzi, M., 2007. Carbohydrate-deficient transferrin (CDT) as a biochemical tool for the screening of congenital disorders of glycosylation (CDGs). Clin. Biochem. 40, 1431–1434. Chantret, I., et al., 2003. A deficiency in dolichyl-P-glucose:Glc1Man9GlcNAc2-PP-dolichyl alpha3-glucosyltransferase defines a new subtype of congenital disorders of glycosylation. J. Biol. Chem. 278, 9962–9971. Couto, J.R., Huffaker, T.C., Robbins, P.W., 1984. Cloning and expression in Escherichia coli of a yeast mannosyltransferase from the asparagine-linked glycosylation pathway. J. Biol. Chem. 259, 378–382. de Koning, T.J., et al., 1998. Recurrent nonimmune hydrops fetalis associated with carbohydrate-deficient glycoprotein syndrome. J. Inherit. Metab. Dis. 21, 681–682. Dohmen, R.J., Strasser, A.W., Höner, C.B., Hollenberg, C.P., 1991. An efficient transformation procedure enabling long-term storage of competent cells of various yeast genera. Yeast 7, 691–692. Dupré, T., et al., 2010. Guanosine diphosphate-mannose:GlcNAc2-PP-dolichol mannosyltransferase deficiency (congenital disorders of glycosylation type Ik): five new patients and seven novel mutations. J. Med. Genet. 47, 729–735. Gao, X.D., Nishikawa, A., Dean, N., 2004. Physical interactions between the Alg1, Alg2, and Alg11 mannosyltransferases of the endoplasmic reticulum. Glycobiology 14, 559–570. Grubenmann, C.E., et al., 2004. Deficiency of the first mannosylation step in the N-glycosylation pathway causes congenital disorder of glycosylation type Ik. Hum. Mol. Genet. 13, 535–542.
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